Angiopoietin-like 4 antibodies and methods of use

ABSTRACT

The present invention relates to monoclonal antibodies binding to human angiopoietin-like 4 protein (hereinafter, sometimes referred to as “ANGPTL4”), and pharmaceutical compositions and methods of treatment comprising the same.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.14/819,680, filed Aug. 6, 2015, which claims priority to U.S.Provisional Patent Application No. 62/034,409, filed Aug. 7, 2014, thecontents of which are incorporated herein by reference in theirentireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which was submittedin ASCII format via EFS-Web on Jan. 26, 2016, in U.S. patent applicationSer. No. 14/819,680, and is hereby incorporated by reference in itsentirety.

BACKGROUND OF THE INVENTION

Angiopoietin-like 4 protein (ANGPTL4) is a member of the angiopoietinlike family of secreted proteins. It is a homooligomeric protein,capable of forming dimers and tetramers, that is expressed by cell typesincluding macrophages, adipose, muscle, and liver cells. ANGPTL4 is alsoknown as hepatic fibrinogen/angiopoietin-related protein (HFARP) (Kim etal. (2000) Biochem. J. 346:603-610); PPAR gamma angiopoietin relatedprotein (PGAR) (Yoon, et al. (2000) Mol. Cell Biol., 20:5343-5349), andfasting induced adipose factor (FIAF) (Kerten et al. (2000) J. Biol.Chem., 275:28488-28493). ANGPTL4 contains an N-terminal coiled-coildomain and a C-terminal fibrinogen (FBN)-like domain (Kim et al. (2000)Biochem. J. 346:603-610).

Lipoprotein lipase (LPL) has a central role in lipoprotein metabolismwhich includes the maintenance of lipoprotein levels in blood and,through tissue specific regulation of its activity. The coiled-coilregion of ANGPTL4 is known to inhibit lipoprotein lipase (LPL)-mediatedtriglyceride (TG) clearance. Therefore, ANGPTL4 loss-of-functionmutations (e.g., as seen in human subjects), genetic deletions (e.g., asseen in transgenic mice), and antibody inhibition (e.g., as seen in miceand cynomolgus monkeys) are all observed to decrease plasmatriglycerides. Furthermore, ANGPTL4 antibodies are also known toactivate LPL. Conversely, ANGPTL4 injection into mice produces a rapidincrease in circulating triglycerides and this is at a higher rate thanthe injection of angiopoietin-like protein 3 (ANGPTL3) (Yoshida et al.(2002) J Lipid Res 43:1770-1772).

The anti-ANGPTL4 antibodies and antigen binding fragments described inthis invention initiate, promote, or enhance activation of LPL, e.g., byblocking ANGPTL4 inhibition of LPL, thereby decreasing plasmatriglycerides. These antibodies are expected to prevent and amelioratethe acute and chronic manifestations of diseases characterized byelevated triglyceride levels, e.g., primary dyslipidemia,hypertriglyceridemia, metabolic syndrome, type II diabetes, and thelike.

SUMMARY OF THE INVENTION

The present invention relates to monoclonal antibodies binding to humanAngiopoietin-like 4 protein (hereinafter, sometimes referred to as“ANGPTL4”), and pharmaceutical compositions and methods of treatmentcomprising the same.

The isolated anti-ANGPTL4 antibodies, or antigen binding fragments,described herein bind ANGPTL4, with an equilibrium dissociation constant(K_(D)) of less than or equal to 100 pM. For example, the isolatedantibodies or antigen binding fragments described herein may bind tohuman ANGPTL4 with a K_(D) of less than or equal to 150 nM, less than orequal to 50 nM, less than or equal to 10 nM, less than or equal to 750pM, less than or equal to 600 pM, less than or equal to 500 pM, lessthan or equal to 400 pM, less than or equal to 300 pM, less than orequal to 200 pM, less than or equal to 100 pM, less than or equal to 75pM, less than or equal to 65 pM, less than or equal to 60 pM, less thanor equal to 55 pM. More specifically, the isolated antibodies or antigenbinding fragments described herein may also bind human ANGPTL4 with aK_(D) of less than or equal to 45 pM, as measured by ForteBio kineticbinding assays, or less than or equal to 24 pM, as measured by solutionequilibrium titration assay (SET); and may also bind cynomolgus monkeyANGPTL4 with a K_(D) of less than or equal to 87 pM, as measured byForteBio kinetic binding assays, or less than or equal to 22 pM, asmeasured by SET.

The present invention relates to an isolated antibody, or antigenbinding fragments thereof, that binds to human ANGPTL4. The presentinvention also relates to an isolated antibody, or antigen bindingfragments thereof, that binds ANGPTL4 and further competes for bindingwith an antibody as described in Table 1. The present invention alsofurther relates to an isolated antibody, or antigen binding fragmentsthereof, that binds the same epitope as an antibody as described inTable 1.

The binding affinity of isolated antibodies and antigen bindingfragments described herein can be determined by solution equilibriumtitration (SET). Methods for SET are known in the art and are describedin further detail below. Alternatively, binding affinity of the isolatedantibodies, or fragments, described herein can be determined by Biacoreassay. Methods for Biacore kinetic assays are known in the art and aredescribed in further detail below.

The isolated anti-ANGPTL4 antibodies and antigen binding fragmentsdescribed herein can be used to inhibit ANGPTL4 binding to lipoproteinlipase (LPL) with an EC₅₀ of less than or equal to 100 nM, less than orequal to 50 nM, less than or equal to 35 nM, less than or equal to 25nM, less than or equal to 10 nM, or less than or equal to 3 nM.

The isolated anti-ANGPTL4 antibodies, or antigen binding fragmentsthereof, may be used to reduce the levels of circulating triglycerides(TG).

The isolated anti-ANGPTL4 antibodies, or antigen binding fragmentsthereof, as described herein can be monoclonal antibodies, human orhumanized antibodies, chimeric antibodies, single chain antibodies, Fabfragments, Fv fragments, F(ab′)2 fragments, or scFv fragments, and/orIgG isotypes.

The isolated anti-ANGPTL4 antibodies, or antigen binding fragmentsthereof, as described herein can also include a framework in which anamino acid has been substituted into the antibody framework from therespective human VH or VL germline sequences.

Another aspect of the invention includes an isolated antibody or antigenbinding fragments thereof having the full heavy and light chainsequences of humanized antibodies described in Table 1. Morespecifically, the isolated antibody or antigen binding fragments thereofcan have the heavy and light chain sequences of NEG276, NEG276-LALA,NEG278, NEG310, NEG313, NEG315, NEG318, NEG319.

A further aspect of the invention includes an isolated antibody orantigen binding fragments thereof having the heavy and light chainvariable domain sequences of humanized antibodies described in Table 1.More specifically, the isolated antibody or antigen binding fragmentthereof can have the heavy and light chain variable domain sequences ofNEG276, NEG276-LALA, NEG278, NEG310, NEG313, NEG315, NEG318, NEG319.

The invention also relates to an isolated antibody or antigen bindingfragments thereof that includes a heavy chain CDR1 selected from thegroup consisting of SEQ ID NOs: 7, 32, 52, 72, 92, 112, and 132; a heavychain CDR2 selected from the group consisting of SEQ ID NOs: 8, 33, 53,73, 93, 113, and 133; and a heavy chain CDR3 selected from the groupconsisting of SEQ ID NOs: 9, 34, 54, 74, 94, 114, and 134, wherein theisolated antibody or antigen binding fragments thereof binds to humanANGPTL4. In another aspect, such isolated antibody or antigen bindingfragments thereof further includes a light chain CDR1 selected from thegroup consisting of SEQ ID NOs: 17, 42, 62, 82, 102, 122, and 142; alight chain CDR2 selected from the group consisting of SEQ ID NOs: 18,43, 63, 83, 103, 123, and 143; and a light chain CDR3 selected from thegroup consisting of SEQ ID NOs: 19, 44, 64, 84, 104, 124, and 144.

The invention also relates to an isolated antibody or antigen bindingfragments thereof that includes a light chain CDR1 selected from thegroup consisting of SEQ ID NOs: 17, 42, 62, 82, 102, 122, and 142; alight chain CDR2 selected from the group consisting of SEQ ID NOs: 18,43, 63, 83, 103, 123, and 143; and a light chain CDR3 selected from thegroup consisting of SEQ ID NOs: 19, 44, 64, 84, 104, 124, and 144,wherein the isolated antibody or antigen binding fragments thereof bindsto human ANGPTL4.

The invention also relates to an isolated antibody or antigen bindingfragments thereof that binds ANGPTL4 having HCDR1, HCDR2, and HCDR3 andLCDR1, LCDR2, and LCDR3, wherein HCDR1, HCDR2, and HCDR3 comprises SEQID NOs: 7, 8, and 9, and LCDR1, LCDR2, LCDR3 comprises SEQ ID NOs: 17,18 and 19; or HCDR1, HCDR2, and HCDR3 comprises SEQ ID NOs: 32, 33, and34 and LCDR1, LCDR2, LCDR3 comprises SEQ ID NOs: 42, 43 and 44; orHCDR1, HCDR2, and HCDR3 comprises SEQ ID NOs: 52, 53, and 54, and LCDR1,LCDR2, LCDR3 comprises SEQ ID NOs: 62, 63, and 64; or HCDR1, HCDR2, andHCDR3 comprises SEQ ID NOs: 72, 73, and 74, and LCDR1, LCDR2, LCDR3comprises SEQ ID NOs: 82, 83, and 84; or HCDR1, HCDR2, and HCDR3comprises SEQ ID NOs: 92, 93, and 94, and LCDR1, LCDR2, LCDR3 comprisesSEQ ID NOs: 102, 103, and 104; or HCDR1, HCDR2, and HCDR3 comprises SEQID NOs: 112, 113, and 114, and LCDR1, LCDR2, LCDR3 comprises SEQ ID NOs:122, 123, and 124; or HCDR1, HCDR2, and HCDR3 comprises SEQ ID NOs: 132,133, and 134, and LCDR1, LCDR2, LCDR3 comprises SEQ ID NOs: 142, 143,and 144.

The invention also relates to an antibody or antigen binding fragmenthaving HCDR1, HCDR2, and HCDR3 of the heavy chain variable domain of SEQID NOs: 13, 38, 58, 78, 98, 118, and 138, and the LCDR1, LCDR2 and LCDR3of the light chain variable domain of SEQ ID NOs: 23, 48, 68, 88, 108,128, and 148, as defined by Chothia. In another aspect of the inventionthe antibody or antigen binding fragment may have the HCDR1, HCDR2, andHCDR3 of the heavy chain variable domain sequence of SEQ ID NOs: 13, 38,58, 78, 98, 118, and 138, and the LCDR1, LCDR2 and LCDR3 of the lightchain variable domain sequence of SEQ ID NOs: 23, 48, 68, 88, 108, 128,and 148, as defined by Kabat.

In one aspect of the invention the isolated antibody or antigen bindingfragments thereof includes a heavy chain variable domain sequenceselected from the group consisting of SEQ ID NOs: 13, 38, 58, 78, 98,118, and 138. The isolated antibody or antigen binding fragment furthercan comprise a light chain variable domain sequence wherein the heavychain variable domain and light chain variable domain combine to formand antigen binding site for ANGPTL4. In particular the light chainvariable domain sequence can be selected from SEQ ID NOs: 23, 48, 68,88, 108, 128, and 148 wherein said isolated antibody or antigen bindingfragments thereof binds ANGPTL4.

The invention also relates to an isolated antibody or antigen bindingfragments thereof that includes a light chain variable domain sequenceselected from the group consisting of SEQ ID NOs: 23, 48, 68, 88, 108,128, and 148, wherein said isolated antibody or antigen bindingfragments thereof binds to human ANGPTL4. The isolated antibody orantigen binding fragment may further comprise a heavy chain variabledomain sequence wherein the light chain variable domain and heavy chainvariable domain combine to form and antigen binding site for ANGPTL4.

In particular, the isolated antibody or antigen binding fragmentsthereof that binds ANGPTL4, may have heavy and light chain variabledomains comprising the sequences of SEQ ID NOs: 13 and 23; 38 and 48; 58and 68; 78 and 88; 98 and 108; 118 and 128; or 138 and 148,respectively.

The invention further relates to an isolated antibody or antigen bindingfragments thereof, that includes a heavy chain variable domain having atleast 90% sequence identity to a sequence selected from the groupconsisting of SEQ ID NOs: 13, 38, 58, 78, 98, 118, and 138, wherein saidantibody binds to ANGPTL4. In one aspect, the isolated antibody orantigen binding fragments thereof also includes a light chain variabledomain having at least 90% sequence identity to a sequence selected fromthe group consisting of SEQ ID NOs: 23, 48, 68, 88, 108, 128, and 148.In a further aspect of the invention, the isolated antibody or antigenbinding fragment has an HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 asdefined by Kabat and as described in Table 1.

The invention also relates to an isolated antibody or antigen bindingfragments thereof, having a light chain variable domain having at least90% sequence identity to a sequence selected from the group consistingof SEQ ID NOs: 23, 48, 68, 88, 108, 128, and 148, wherein said antibodybinds ANGPTL4.

In another aspect of the invention, the isolated antibody, or antigenbinding fragments thereof, that binds to ANGPTL4 may have a heavy chaincomprising the sequence of SEQ ID NOs: 15, 28, 40, 60, 80, 100, 120, and140. The isolated antibody can also includes a light chain that cancombine with the heavy chain to form an antigen binding site to humanANGPTL4. In particular, the light chain may have a sequence comprisingSEQ ID NOs: 25, 50, 70, 90, 110, 130, and 150. In particular, theisolated antibody or antigen binding fragments thereof that bindsANGPTL4, may have a heavy chain and a light chain comprising thesequences of SEQ ID NOs: 15 and 25; 28 and 25; 40 and 50; 60 and 70; 80and 90; 100 and 110; 120 and 130; or 140 and 150, respectively.

The invention still further relates to an isolated antibody or antigenbinding fragments thereof that includes a heavy chain having at least90% sequence identity to a sequence selected from the group consistingof SEQ ID NOs: 15, 28, 40, 60, 80, 100, 120, and 140, wherein saidantibody binds to ANGPTL4. In one aspect, the isolated antibody orantigen binding fragments thereof also includes a light chain having atleast 90% sequence identity to a sequence selected from the groupconsisting of SEQ ID NOs: 25, 50, 70, 90, 110, 130, and 150.

The invention still further relates to an isolated antibody or antigenbinding fragments thereof that includes a light chain having at least90% sequence identity to a sequence selected from the group consistingof SEQ ID NOs: 25, 50, 70, 90, 110, 130, and 150, wherein said antibodybinds ANGPTL4.

The invention still further relates to an isolated antibody or antigenbinding fragment which competes for binding with the antibodies orantigen binding fragments described herein, e.g., with humanizedantibodies NEG276, NEG276-LALA, NEG278, NEG310, NEG313, NEG315, NEG318,and NEG319. In one embodiment, the isolated antibody or antigen bindingfragment of the invention is capable of inhibiting by more than 50% thebinding of ANGPTL4 by a humanized antibody selected from NEG276,NEG276-LALA, NEG278, NEG310, NEG313, NEG315, NEG318, and NEG319, whenthe two antibodies or antigen binding fragments are present in equimolarconcentrations.

In another embodiment, the isolated antibody or antigen binding fragmentof the invention is capable of inhibiting by more than 80% the bindingof ANGPTL4 by a humanized antibody selected from NEG276, NEG276-LALA,NEG278, NEG310, NEG313, NEG315, NEG318, and NEG319, when the twoantibodies or antigen binding fragments are present in equimolarconcentrations. In still other embodiments, the isolated antibody orantigen binding fragment of the invention is capable of inhibiting bymore than 85% (or 90%, 95%, 98% or 99%) the binding of ANGPTL4 by ahumanized antibody selected from NEG276, NEG276-LALA, NEG278, NEG310,NEG313, NEG315, NEG318, and NEG319, when the two antibodies or antigenbinding fragments are present in equimolar concentrations.

The invention also relates to compositions comprising the isolatedantibody, or antigen binding fragments thereof, described herein. Aswell as, antibody compositions in combination with a pharmaceuticallyacceptable carrier. Specifically, the invention further includespharmaceutical compositions comprising an antibody or antigen bindingfragments thereof of Table 1, such as, for example humanized antibodiesNEG276, NEG276-LALA, NEG278, NEG310, NEG313, NEG315, NEG318, NEG319. Theinvention also relates to pharmaceutical compositions comprising acombination of two or more of the isolated antibodies or antigen bindingfragments thereof of Table 1.

The invention also relates to an isolated nucleic acid sequence encodingthe heavy chain variable domain having a sequence selected from SEQ IDNOs: 13, 38, 58, 78, 98, 118, and 138. In particular the nucleic acidhas a sequence at least 90% sequence identity to a sequence selectedfrom the group consisting of SEQ ID NOs: 14, 27, 39, 59, 79, 99, 119,and 139. In a further aspect of the invention the sequence is SEQ IDNOs: 14, 27, 39, 59, 79, 99, 119, or 139.

The invention also relates to an isolated nucleic acid sequence encodingthe light chain variable domain having a sequence selected from SEQ IDNOs: 23, 48, 68, 88, 108, 128, and 148. In particular the nucleic acidhas a sequence at least 90% sequence identity to a sequence selectedfrom the group consisting of SEQ ID NOs: 24, 31, 49, 69, 89, 109, 129,and 149. In a further aspect of the invention the sequence is SEQ IDNOs: 24, 31, 49, 69, 89, 109, 129, or 149.

The invention also relates to an isolated nucleic acid comprising asequence encoding a polypeptide that includes a light chain variabledomain having at least 90% sequence identity to a sequence selected fromthe group consisting of SEQ ID NOs: 23, 48, 68, 88, 108, 128, and 148.

The invention also relates to a vector that includes one or more of thenucleic acid molecules described herein.

The invention also relates to an isolated host cell that includes arecombinant DNA sequence encoding a heavy chain of the antibodydescribed above, and a second recombinant DNA sequence encoding a lightchain of the antibody described above, wherein said DNA sequences areoperably linked to a promoter and are capable of being expressed in thehost cell. It is contemplated that the antibody can be a humanizedantibody. It is also contemplated that the host cell is a non-humanmammalian cell.

It is contemplated that the cell is a human cell. It is furthercontemplated that the cell is in a subject. In one embodiment, it iscontemplated that the cell is an endothelial cell. In other embodiments,the cell may be one or more of adipose, muscle, and liver cells. It isstill further contemplated that the subject is human.

The invention also relates to a method of treating, improving, orpreventing a ANGPTL4-associated disorder in a patient, wherein themethod includes the step of administering to the patient an effectiveamount of a composition comprising the antibody or antigen bindingfragments thereof described herein. In one aspect, theANGPTL4-associated disorder is associated with hypertriglyceridemia(e.g., severe hypertriglyceridemia (e.g., with plasma triglycerideconcentration >500 mg/dL), hypertriglyceridemia associated with obesity,and type V hypertriglyceridemia). In other aspects, theANGPTL4-associated disorder is associated with primary dyslipidemia,metabolic syndrome, type II diabetes. It is contemplated that thepatient is human.

Any of the foregoing isolated antibodies or antigen binding fragmentsthereof may be a monoclonal antibody or antigen binding fragmentsthereof.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which this invention pertains.

The terms “ANGPTL4 protein” or “ANGPTL4 antigen” or “ANGPTL4” are usedinterchangeably, and refer to the Angiopoietin-like 4 (ANGPTL4) proteinin different species. For example, human ANGPTL4 has the sequence as setout in Table 1 (SEQ ID NO: 1), and has been described in previousreports and literature (Nature, Vol. 386, p. 73-77, 1997; Genomics, Vol.54, No. 2, p. 191-199, 1998; Biochem. J., Vol. 339, Part 1, P. 177-184,1999; Genbank Accession No. NP 002534). ANGPTL4 contains an N-terminalcoiled-coil domain and a C-terminal fibrinogen (FBN)-like domain (Kim etal. (2000) Biochem. J. 346:603-610). It is a homooligomeric protein,capable of forming dimers and tetramers, that is expressed by cell typesincluding macrophages, adipose, muscle, and liver cells, and known toinhibit lipoprotein lipase (LPL)-mediated triglyceride (TG) clearance.

In addition, in the context of this invention, the term “ANGPTL4”includes mutants of the natural Angiopoietin-like 4 (ANGPTL4) protein,which have substantially the same amino acid sequence as that of thenative primary structure (amino acid sequence) described in theabove-mentioned reports. Herein, the term “mutants of the natural humanAngiopoietin-like 4 (ANGPTL4) protein having substantially the sameamino acid sequence” refers to such mutant proteins.

The term “antibody” as used herein means a whole antibody and anyantigen binding fragment (i.e., “antigen-binding portion”) or singlechain thereof. A whole antibody is a glycoprotein comprising at leasttwo heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds. Each heavy chain is comprised of a heavy chain variableregion (abbreviated herein as VH) and a heavy chain constant region. Theheavy chain constant region is comprised of three domains, CH1, CH2 andCH3. Each light chain is comprised of a light chain variable region(abbreviated herein as VL) and a light chain constant region. The lightchain constant region is comprised of one domain, CL. The VH and VLregions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). Each VHand VL is composed of three CDRs and four FRs arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system.

The term “antigen binding portion” or “antigen binding fragment” of anantibody, as used herein, refers to one or more fragments of an intactantibody that retain the ability to specifically bind to a given antigen(e.g., human oxidized LDL receptor (ANGPTL4)). Antigen binding functionsof an antibody can be performed by fragments of an intact antibody.Examples of binding fragments encompassed within the term antigenbinding portion or antigen binding fragment of an antibody include a Fabfragment, a monovalent fragment consisting of the VL, VH, CL and CH1domains; a F(ab)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; an Fdfragment consisting of the VH and CH1 domains; an Fv fragment consistingof the VL and VH domains of a single arm of an antibody; a single domainantibody (dAb) fragment (Ward et al., 1989 Nature 341:544-546), whichconsists of a VH domain or a VL domain; and an isolated complementaritydetermining region (CDR).

Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by an artificial peptide linker that enables them to be made asa single protein chain in which the VL and VH regions pair to formmonovalent molecules (known as single chain Fv (scFv); see, e.g., Birdet al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl.Acad. Sci. 85:5879-5883). Such single chain antibodies include one ormore antigen binding portions or fragments of an antibody. Theseantibody fragments are obtained using conventional techniques known tothose of skill in the art, and the fragments are screened for utility inthe same manner as are intact antibodies.

Antigen binding fragments can also be incorporated into single domainantibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies,tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, 2005,Nature Biotechnology, 23, 9, 1126-1136). Antigen binding portions ofantibodies can be grafted into scaffolds based on polypeptides such asFibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describesfibronectin polypeptide monobodies).

Antigen binding fragments can be incorporated into single chainmolecules comprising a pair of tandem Fv segments (VH—CH1-VH—CH1) which,together with complementary light chain polypeptides, form a pair ofantigen binding regions (Zapata et al., 1995 Protein Eng.8(10):1057-1062; and U.S. Pat. No. 5,641,870).

As used herein, the term “affinity” refers to the strength ofinteraction between antibody and antigen at single antigenic sites.Within each antigenic site, the variable region of the antibody “arm”interacts through weak non-covalent forces with antigen at numeroussites; the more interactions, the stronger the affinity. As used herein,the term “high affinity” for an antibody or antigen binding fragmentsthereof (e.g., a Fab fragment) generally refers to an antibody, orantigen binding fragment, having a KD of 10⁻⁹M or less.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refer to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an alpha carbon that is boundto a hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

The term “binding specificity” as used herein refers to the ability ofan individual antibody combining site to react with only one antigenicdeterminant.

The phrase “specifically (or selectively) binds” to an antibody (e.g., aANGPTL4-binding antibody) refers to a binding reaction that isdeterminative of the presence of a cognate antigen (e.g., a humanANGPTL4 or cynomolgus ANGPTL4) in a heterogeneous population of proteinsand other biologics. The phrases “an antibody recognizing an antigen”and “an antibody specific for an antigen” are used interchangeablyherein with the term “an antibody which binds specifically to anantigen.”

The term “ANGPTL4 mediated” refers to the fact that ANGPTL4 is known toinhibit lipoprotein lipase (LPL)-mediated triglyceride (TG) clearance,and thereby increase triglyceride levels.

An “ANGPTL4-associated disorder,” “ANGPTL4-associated condition,” orsimilar terms as used herein, refer to any number of conditions ordiseases in which ANGPTL4ANGPTL4 a reduction of ANGPTL4-mediated LPLinhibition and lipoprotein modulation is sought. These conditionsinclude but are not limited to those involving lipid metabolism, such ashyperlipidemia, hyperlipoproteinemia and dyslipidemia, includingatherogenic dyslipidemia, diabetic dyslipidemia, hypertriglyceridemia(e.g., severe hypertriglyceridemia (e.g., with plasma triglycerideconcentration >500 mg/dL), hypertriglyceridemia associated with obesity,and type V hypertriglyceridemia), hypercholesterolemia, chylomicronemia,mixed dyslipidemia (obesity, metabolic syndrome, diabetes, etc.),lipodystrophy, lipoatrophy, and other conditions caused by, e.g.,decreased LPL activity and/or LPL deficiency, decreased LDL receptoractivity and/or LDL receptor deficiency, altered ApoC2, ApoE deficiency,increased ApoB, increased production and/or decreased elimination ofvery low-density lipoprotein (VLDL), certain drug treatment (e.g.,glucocorticoid treatment-induced dyslipidemia), any geneticpredisposition, diet, life style, and the like.

Other ANGPTL4-associated diseases or disorders associated with orresulting from hyperlipidemia, hyperlipoproteinemia, and/ordyslipidemia, include, but are not limited to, cardiovascular diseasesor disorders, such as atherosclerosis, aneurysm, hypertension, angina,stroke, cerebrovascular diseases, congestive heart failure, coronaryartery diseases, myocardial infarction, peripheral vascular diseases,and the like; acute pancreatitis; nonalcoholic steatohepatitis (NASH);blood sugar disorders, such as diabetes; obesity, and the like.

The term “chimeric antibody” is an antibody molecule in which (a) theconstant region, or a portion thereof, is altered, replaced or exchangedso that the antigen binding site (variable region) is linked to aconstant region of a different or altered class, effector functionand/or species, or an entirely different molecule which confers newproperties to the chimeric antibody, e.g., an enzyme, toxin, hormone,growth factor, drug, etc.; or (b) the variable region, or a portionthereof, is altered, replaced or exchanged with a variable region havinga different or altered antigen specificity. For example, a mouseantibody can be modified by replacing its constant region with theconstant region from a human immunoglobulin. Due to the replacement witha human constant region, the chimeric antibody can retain itsspecificity in recognizing the antigen while having reduced antigenicityin human as compared to the original mouse antibody.

The term “conservatively modified variant” applies to both amino acidand nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidthat encodes a polypeptide is implicit in each described sequence.

For polypeptide sequences, “conservatively modified variants” includeindividual substitutions, deletions or additions to a polypeptidesequence which result in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of theinvention. The following eight groups contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)). In someembodiments, the term “conservative sequence modifications” are used torefer to amino acid modifications that do not significantly affect oralter the binding characteristics of the antibody containing the aminoacid sequence.

The term “epitope” means a protein determinant capable of specificbinding to an antibody. Epitopes usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. Conformational andnonconformational epitopes are distinguished in that the binding to theformer but not the latter is lost in the presence of denaturingsolvents.

The term “human antibody”, as used herein, is intended to includeantibodies having variable regions in which both the framework and CDRregions are derived from sequences of human origin. Furthermore, if theantibody contains a constant region, the constant region also is derivedfrom such human sequences, e.g., human germline sequences, or mutatedversions of human germline sequences. The human antibodies of theinvention may include amino acid residues not encoded by human sequences(e.g., mutations introduced by random or site-specific mutagenesis invitro or by somatic mutation in vivo).

A “humanized” antibody is an antibody that retains the antigen-specificreactivity of a non-human antibody, e.g., a mouse monoclonal antibody,while being less immunogenic when administered as a therapeutic inhumans. See, e.g., Robello et al., Transplantation, 68: 1417-1420. Thiscan be achieved, for instance, by retaining the non-humanantigen-binding regions and replacing the remaining parts of theantibody with their human counterparts (i.e., the constant region aswell as portions of the variable region not involved in binding). See,e.g., Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855, 1984;Morrison and Oi, Adv. Immunol., 44:65-92, 1989; Verhoeyen et al.,Science, 239:1534-1536, 1988; Padlan, Molec. Immun., 28:489-498, 1991;and Padlan, Molec. Immun., 31:169-217, 1994. Other examples of humanengineering technology include, but are not limited to Xoma technologydisclosed in U.S. Pat. No. 5,766,886.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same. Two sequences are“substantially identical” if two sequences have a specified percentageof amino acid residues or nucleotides that are the same (i.e., 60%identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identityover a specified region, or, when not specified, over the entiresequence), when compared and aligned for maximum correspondence over acomparison window, or designated region as measured using one of thefollowing sequence comparison algorithms or by manual alignment andvisual inspection. Optionally, the identity exists over a region that isat least about 50 nucleotides (or 10 amino acids) in length, or morepreferably over a region that is 100 to 500 or 1000 or more nucleotides(or 20, 50, 200 or more amino acids) in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homologyalignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443, 1970,by the search for similarity method of Pearson and Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444, 1988, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection (see, e.g., Brent etal., Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(Ringbou ed., 2003)).

Two examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al., Nuc. Acids Res.25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410,1990, respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information.This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,supra). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are extendedin both directions along each sequence for as far as the cumulativealignment score can be increased. Cumulative scores are calculatedusing, for nucleotide sequences, the parameters M (reward score for apair of matching residues; always >0) and N (penalty score formismatching residues; always <0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T, and Xdetermine the sensitivity and speed of the alignment. The BLASTN program(for nucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) or 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989)alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, Proc.Natl. Acad. Sci. USA 90:5873-5787, 1993). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The percent identity between two amino acid sequences can also bedetermined using the algorithm of E. Meyers and W. Miller (Comput. Appl.Biosci., 4:11-17, 1988) which has been incorporated into the ALIGNprogram (version 2.0), using a PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4. In addition, the percent identitybetween two amino acid sequences can be determined using the Needlemanand Wunsch (J. Mol, Biol. 48:444-453, 1970) algorithm which has beenincorporated into the GAP program in the GCG software package (availableon the world wide web at gcg.com), using either a Blossom 62 matrix or aPAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and alength weight of 1, 2, 3, 4, 5, or 6.

Other than percentage of sequence identity noted above, anotherindication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

The term “isolated antibody” refers to an antibody that is substantiallyfree of other antibodies having different antigenic specificities (e.g.,an isolated antibody that specifically binds ANGPTL4 is substantiallyfree of antibodies that specifically bind antigens other than ANGPTL4).An isolated antibody that specifically binds ANGPTL4 may, however, havecross-reactivity to other antigens. Moreover, an isolated antibody maybe substantially free of other cellular material and/or chemicals.

The term “isotype” refers to the antibody class (e.g., IgM, IgE, IgGsuch as IgG1 or IgG4) that is provided by the heavy chain constantregion genes. Isotype also includes modified versions of one of theseclasses, where modifications have been made to alter the Fc function,for example, to enhance or reduce effector functions or binding to Fcreceptors.

The term “k_(assoc)” or “k_(a)”, as used herein, is intended to refer tothe association rate of a particular antibody-antigen interaction,whereas the term “k_(dis)” or “k_(d),” as used herein, is intended torefer to the dissociation rate of a particular antibody-antigeninteraction. The term “K_(D)”, as used herein, is intended to refer tothe dissociation constant, which is obtained from the ratio of k_(d) tok_(a) (i.e. k_(d)/k_(a)) and is expressed as a molar concentration (M).K_(D) values for antibodies can be determined using methods wellestablished in the art. Methods for determining the K_(D) of an antibodyinclude measuring surface plasmon resonance using a biosensor systemsuch as a Biacore® system, or measuring affinity in solution by solutionequilibrium titration (SET).

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “nucleic acid” is used herein interchangeably with the term“polynucleotide” and refers to deoxyribonucleotides or ribonucleotidesand polymers thereof in either single- or double-stranded form. The termencompasses nucleic acids containing known nucleotide analogs ormodified backbone residues or linkages, which are synthetic, naturallyoccurring, and non-naturally occurring, which have similar bindingproperties as the reference nucleic acid, and which are metabolized in amanner similar to the reference nucleotides. Examples of such analogsinclude, without limitation, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides,peptide-nucleic acids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, as detailed below,degenerate codon substitutions may be achieved by generating sequencesin which the third position of one or more selected (or all) codons issubstituted with mixed-base and/or deoxyinosine residues (Batzer et al.,Nucleic Acid Res. 19:5081, 1991; Ohtsuka et al., J. Biol. Chem.260:2605-2608, 1985; and Rossolini et al., Mol. Cell. Probes 8:91-98,1994).

The term “operably linked” refers to a functional relationship betweentwo or more polynucleotide (e.g., DNA) segments. Typically, the termrefers to the functional relationship of a transcriptional regulatorysequence to a transcribed sequence. For example, a promoter or enhancersequence is operably linked to a coding sequence if it stimulates ormodulates the transcription of the coding sequence in an appropriatehost cell or other expression system. Generally, promotertranscriptional regulatory sequences that are operably linked to atranscribed sequence are physically contiguous to the transcribedsequence, i.e., they are cis-acting. However, some transcriptionalregulatory sequences, such as enhancers, need not be physicallycontiguous or located in close proximity to the coding sequences whosetranscription they enhance.

As used herein, the term, “optimized” means that a nucleotide sequencehas been altered to encode an amino acid sequence using codons that arepreferred in the production cell or organism, generally a eukaryoticcell, for example, a cell of Pichia, a Chinese Hamster Ovary cell (CHO)or a human cell. The optimized nucleotide sequence is engineered toretain completely or as much as possible the amino acid sequenceoriginally encoded by the starting nucleotide sequence, which is alsoknown as the “parental” sequence. The optimized sequences herein havebeen engineered to have codons that are preferred in mammalian cells.However, optimized expression of these sequences in other eukaryoticcells or prokaryotic cells is also envisioned herein. The amino acidsequences encoded by optimized nucleotide sequences are also referred toas optimized.

The terms “polypeptide” and “protein” are used interchangeably herein torefer to a polymer of amino acid residues. The terms apply to amino acidpolymers in which one or more amino acid residue is an artificialchemical mimetic of a corresponding naturally occurring amino acid, aswell as to naturally occurring amino acid polymers and non-naturallyoccurring amino acid polymer. Unless otherwise indicated, a particularpolypeptide sequence also implicitly encompasses conservatively modifiedvariants thereof.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as antibodies isolated from an animal (e.g., amouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom, antibodies isolated from a hostcell transformed to express the human antibody, e.g., from atransfectoma, antibodies isolated from a recombinant, combinatorialhuman antibody library, and antibodies prepared, expressed, created orisolated by any other means that involve splicing of all or a portion ofa human immunoglobulin gene, sequences to other DNA sequences. Suchrecombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the VH and VL regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline VH and VL sequences, may not naturally existwithin the human antibody germline repertoire in vivo.

The term “recombinant host cell” (or simply “host cell”) refers to acell into which a recombinant expression vector has been introduced. Itshould be understood that such terms are intended to refer not only tothe particular subject cell but to the progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term “host cell” as used herein.

The term “subject” includes human and non-human animals. Non-humananimals include all vertebrates (e.g.: mammals and non-mammals) such as,non-human primates (e.g.: cynomolgus monkey), sheep, dog, cow, chickens,amphibians, and reptiles. Except when noted, the terms “patient” or“subject” are used herein interchangeably. As used herein, the terms“cyno” or “cynomolgus” refer to the cynomolgus monkey (Macacafascicularis).

As used herein, the term “treating” or “treatment” of any disease ordisorder (e.g., ANGPTL4 associated disorder) refers in one embodiment,to ameliorating the disease or disorder (i.e., slowing or arresting orreducing the development of the disease or at least one of the clinicalsymptoms thereof). In another embodiment “treating” or “treatment”refers to alleviating or ameliorating at least one physical parameterincluding those which may not be discernible by the patient. In yetanother embodiment, “treating” or “treatment” refers to modulating thedisease or disorder, either physically, (e.g., stabilization of adiscernible symptom), physiologically, (e.g., stabilization of aphysical parameter), or both. In yet another embodiment, “treating” or“treatment” refers to preventing or delaying the onset or development orprogression of the disease or disorder.

“Prevention” as it relates to indications described herein, including,e.g., ANGPTL4 associated disorder, means any action that prevents orslows a worsening in e.g., ANGPTL4 associated disease parameters, asdescribed below, in a patient at risk for said worsening.

The term “vector” is intended to refer to a polynucleotide moleculecapable of transporting another polynucleotide to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments may beligated. Another type of vector is a viral vector, such as anadeno-associated viral vector (AAV, or AAV2), wherein additional DNAsegments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D depicts the reversal of ANGPTL4-mediated inhibition of humanlipoprotein lipase (LPL) protein by selected ANGPTL4 antibodies of theinvention.

FIG. 2 depicts binding of selected antibodies of the invention tofull-length human ANGPTL4 and human ANGPTL4 N-terminal coiled coildomain, and absence of binding to human full-length ANGPTL3. ANGPTL3Ab=ANGPTL3-specific reference antibody.

FIG. 3A-3B depicts changes in plasma triglyceride levels in humanANGPTL4 transgenic mice following administration of selected ANGPTL4antibodies of the invention.

FIG. 4 depicts plasma total human antibody concentrations in obese,diabetic cynomolgus monkeys following administration of one ANGPTL4antibody of the invention (NEG276-LALA).

FIG. 5 depicts changes in plasma triglyceride (TG) concentrations inobese, diabetic cynomolgus monkeys following administration of oneANGPTL4 antibody of the invention (NEG276-LALA).

FIG. 6 depicts changes in plasma total cholesterol concentration inobese, diabetic cynomolgus monkeys following administration of oneANGPTL4 antibody of the invention (NEG276-LALA).

FIG. 7 depicts changes in plasma high-density lipoprotein (HDL)concentrations in obese, diabetic cynomolgus monkeys followingadministration of one ANGPTL4 antibody of the invention (NEG276-LALA).

FIG. 8 depicts changes in plasma total apolipoprotein B (ApoB)concentrations in obese, diabetic cynomolgus monkeys followingadministration of one ANGPTL4 antibody of the invention (NEG276-LALA).

FIG. 9 depicts changes in plasma apolipoprotein C-III (ApoC-III)concentrations in obese, diabetic cynomolgus monkeys followingadministration of one ANGPTL4 antibody of the invention (NEG276-LALA).

FIG. 10 depicts changes in plasma lipoprotein-associated cholesterollevels as assessed by fast-protein liquid chromatography (FPLC)separation of plasma lipoprotein following administration of one ANGPTL4antibody of the invention. Data from one monkey is shown (NEG276-LALA,monkey #6296). Abbreviations: TRL, triglyceride-rich lipoproteins; LDL,low-density lipoprotein; HDL, high-density lipoprotein.

FIG. 11 depicts changes in plasma lipoprotein-associated triglyceride(TG) levels as assessed by fast-protein liquid chromatography (FPLC)separation of plasma lipoprotein following administration of one ANGPTL4antibody of the invention. Data from one monkey is shown (NEG276-LALA,monkey #6296). Abbreviations: TRL, triglyceride-rich lipoproteins; LDL,low-density lipoprotein; HDL, high-density lipoprotein.

DETAILED DESCRIPTION

The present invention is based, in part, on the discovery of antibodymolecules that specifically bind to ANGPTL4 and inhibit its biologicalactivities. The invention relates to both full IgG format antibodies(e.g., humanized antibodies NEG276, NEG276-LALA, NEG278, NEG310, NEG313,NEG315, NEG318, NEG319) as well as antigen binding fragments thereof,such as Fab fragments.

Accordingly, the present invention provides antibodies that specificallybind to ANGPTL4 (e.g., human ANGPTL4), pharmaceutical compositions,production methods, and methods of use of such antibodies andcompositions.

ANGPTL4 Proteins

The present invention provides antibodies that specifically bind toANGPTL4 and inhibit its biological activities, including ability toactivate lipoprotein lipase (LPL). Conversely,

Angiopoietin-like 4 protein (ANGPTL4) is a member of the angiopoietinfamily of secreted proteins. It is a homooligomeric protein, capable offorming dimers and tetramers, that is expressed by cell types includingmacrophages, adipose, muscle, and liver cells. ANGPTL4 is also known ashepatic fibrinogen/angiopoietin-related protein (HFARP)(Kim et al.(2000) Biochem. J. 346:603-610); PPAR gamma angiopoietin related protein(PGAR)(Yoon, et al. (2000) Mol. Cell Biol., 20:5343-5349), and fastinginduced adipose factor (FIAF)(Kerten et al. (2000) J. Biol. Chem.,275:28488-28493). ANGPTL4 contains an N-terminal coiled-coil domain anda C-terminal fibrinogen (FBN)-like domain (Kim et al. (2000) Biochem. J.346:603-610).

Lipoprotein lipase (LPL) has a central role in lipoprotein metabolism tomaintain normal lipoprotein levels in blood and, through tissue specificregulation of its activity, to determine when and in what tissuestriglycerides (TG) are unloaded. The coiled-coil region of ANGPTL4 isknown to inhibit lipoprotein lipase (LPL)-mediated triglyceride (TG)clearance. Therefore, ANGPTL4 loss-of-function mutations (e.g., as seenin human subjects), genetic deletions (e.g., as seen in transgenicmice), and antibody inhibition (e.g., as seen in mice and cynomolgusmonkeys) are all observed to decrease plasma triglycerides. Furthermore,ANGPTL4 antibodies are also known to activate LPL. Conversely, ANGPTL4injection into mice produces a rapid increase in circulatingtriglycerides and this is at a higher rate than the injection ofangiopoietin-like protein 3 (ANGPTL3) (Yoshida et al. (2002) J Lipid Res43:1770-1772).

The anti-ANGPTL4 antibodies and antigen binding fragments described inthis invention initiate, promote, or enhance activation of LPL, e.g., byblocking ANGPTL4 inhibition of LPL, thereby decreasing plasmatriglycerides. These antibodies are expected to prevent and amelioratethe acute and chronic manifestations of diseases characterized byelevated triglyceride levels, e.g., primary dyslipidemia,hypertriglyceridemia, metabolic syndrome, type II diabetes, and thelike.

The anti-ANGPTL4 antibodies and antigen binding fragments described inthis invention initiate, promote, or enhance activation of LPL, e.g., byblocking ANGPTL4 inhibition of LPL, thereby decreasing plasmatriglycerides. These antibodies are expected to prevent and amelioratethe acute and chronic manifestations of diseases characterized byelevated triglyceride levels, e.g., primary dyslipidemia,hypertriglyceridemia, metabolic syndrome, type II diabetes, and thelike.

ANGPTL4 Antibodies & Antigen Binding Fragments

The present invention provides antibodies that specifically bind toANGPTL4. In some embodiments, the present invention provides antibodiesthat specifically bind to human and cynomolgus monkey ANGPTL4.Antibodies of the invention include, but are not limited to, thehumanized antibodies and Fabs, isolated as described in the Examples.

The present invention provides antibodies that specifically bind aANGPTL4 protein (e.g., human and cynomolgus monkey ANGPTL4), wherein theantibodies comprise a VH domain having an amino acid sequence of SEQ IDNOs: 13, 38, 58, 78, 98, 118, and 138. The present invention alsoprovides antibodies that specifically bind to a ANGPTL4 protein, whereinthe antibodies comprise a VH CDR having an amino acid sequence of anyone of the VH CDRs listed in Table 1, infra. In particular, theinvention provides antibodies that specifically bind to an ANGPTL4protein (e.g., human and cynomolgus monkey ANGPTL4), wherein theantibodies comprise (or alternatively, consist of) one, two, three, ormore VH CDRs having an amino acid sequence of any of the VH CDRs listedin Table 1, infra.

The present invention provides antibodies that specifically bind to aANGPTL4 protein, said antibodies comprising a VL domain having an aminoacid sequence of SEQ ID NOs: 23, 48, 68, 88, 108, 128, and 148. Thepresent invention also provides antibodies that specifically bind to anANGPTL4 protein (e.g., human and cynomolgus monkey ANGPTL4), saidantibodies comprising a VL CDR having an amino acid sequence of any oneof the VL CDRs listed in Table 2, infra. In particular, the inventionprovides antibodies that specifically bind to an ANGPTL4 protein (e.g.,human and cynomolgus monkey ANGPTL4), said antibodies comprising (oralternatively, consisting of) one, two, three or more VL CDRs having anamino acid sequence of any of the VL CDRs listed in Table 1, infra.

Other antibodies of the invention include amino acids that have beenmutated, yet have at least 60, 70, 80, 85, 90 or 95 percent identity inthe CDR regions with the CDR regions depicted in the sequences describedin Table 1. In some embodiments, it includes mutant amino acid sequenceswherein no more than 1, 2, 3, 4 or 5 amino acids have been mutated inthe CDR regions when compared with the CDR regions depicted in thesequence described in Table 1.

The present invention also provides nucleic acid sequences that encodeVH, VL, the full-length heavy chain, and the full-length light chain ofthe antibodies that specifically bind to an ANGPTL4 protein (e.g., humanand cynomolgus monkey ANGPTL4). Such nucleic acid sequences can beoptimized for expression in mammalian cells (for example, Table 1 showsthe optimized nucleic acid sequences for the heavy chain and light chainof antibodies of the invention).

TABLE 1 Examples of ANGPTL4 Antibodies, Fabs and ANGPTL4 ProteinsSequence Sequence Identifier Description (SEQ ID NO.)Amino acid or polynucleotide sequence Human ANGPTL4   1MSGAPTAGAALMLCAATAVLLSAQGGPVQSKSPRFASWDEMN amino acidVLAHGLLQLGQGLREHAERTRSQLSALERRLSACGSACQGTE sequence (NCBIGSTDLPLAPESRVDPEVLHSLQTQLKAQNSRIQQLFHKVAQQ ReferenceQRHLEKQHLRIQHLQSQFGLLDHKHLDHEVAKPARRKRLPEM Sequence:AQPVDPAHNVSRLHRLPRDCQELFQVGERQSGLFEIQPQGSP NM_139314.2)PFLVNCKMTSDGGWTVIQRRHDGSVDFNRPWEAYKAGFGDPHGEFWLGLEKVHSITGDRNSRLAVQLRDWDGNAELLQFSVHLGGEDTAYSLQLTAPVAGQLGATTVPPSGLSVPFSTWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNLNGQYFRSIPQQRQKLKKGI FWKTWRGRYYPLQATTMLIQPMAAEAASHuman ANGPTL4   2 ATGAGCGGTGCTCCGACGGCCGGGGCAGCCCTGATGCTCTGCnucleic acid GCCGCCACCGCCGTGCTACTGAGCGCTCAGGGCGGACCCGTG sequence (NCBICAGTCCAAGTCGCCGCGCTTTGCGTCCTGGGACGAGATGAAT ReferenceGTCCTGGCGCACGGACTCCTGCAGCTCGGCCAGGGGCTGCGC NM_139314.2)GAACACGCGGAGCGCACCCGCAGTCAGCTGAGCGCGCTGGAGCGGCGCCTGAGCGCGTGCGGGTCCGCCTGTCAGGGAACCGAGGGGTCCACCGACCTCCCGTTAGCCCCTGAGAGCCGGGTGGACCCTGAGGTCCTTCACAGCCTGCAGACACAACTCAAGGCTCAGAACAGCAGGATCCAGCAACTCTTCCACAAGGTGGCCCAGCAGCAGCGGCACCTGGAGAAGCAGCACCTGCGAATTCAGCATCTGCAAAGCCAGTTTGGCCTCCTGGACCACAAGCACCTAGACCATGAGGTGGCCAAGCCTGCCCGAAGAAAGAGGCTGCCCGAGATGGCCCAGCCAGTTGACCCGGCTCACAATGTCAGCCGCCTGCACCGGCTGCCCAGGGATTGCCAGGAGCTGTTCCAGGTTGGGGAGAGGCAGAGTGGACTATTTGAAATCCAGCCTCAGGGGTCTCCGCCATTTTTGGTGAACTGCAAGATGACCTCAGATGGAGGCTGGACAGTAATTCAGAGGCGCCACGATGGCTCAGTGGACTTCAACCGGCCCTGGGAAGCCTACAAGGCGGGGTTTGGGGATCCCCACGGCGAGTTCTGGCTGGGTCTGGAGAAGGTGCATAGCATCACGGGGGACCGCAACAGCCGCCTGGCCGTGCAGCTGCGGGACTGGGATGGCAACGCCGAGTTGCTGCAGTTCTCCGTGCACCTGGGTGGCGAGGACACGGCCTATAGCCTGCAGCTCACTGCACCCGTGGCCGGCCAGCTGGGCGCCACCACCGTCCCACCCAGCGGCCTCTCCGTACCCTTCTCCACTTGGGACCAGGATCACGACCTCCGCAGGGACAAGAACTGCGCCAAGAGCCTCTCTGGAGGCTGGTGGTTTGGCACCTGCAGCCATTCCAACCTCAACGGCCAGTACTTCCGCTCCATCCCACAGCAGCGGCAGAAGCTTAAGAAGGGAATCTTCTGGAAGACCTGGCGGGGCCGCTACTACCCGCTGCAGGCCACCACCATGTTGATCCAGCCCATGGCAGCAGAGGCAGCCTCC TAGCGTC Cyno ANGPTL4   3MRGAPTAGAALMLCVATAVLLRAQGGPVQSKSPRFASWDEMN (amino acidVLAHGLLQLGQGLREHAERTRSQLNALERRLSACGSACQGTE sequence)GSTALPLAPESRVDPEVLHSLQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLRIQRLQSQVGLLDPKHLDHEVAKPARRKRRPEMAQPVDSAHNASRLHRLPRDCQELFEDGERQSGLFEIQPQGSPPFLVNCKMTSDGGWTVIQRRHDGSVDFNRPWEAYKAGFGDPQGEFWLGLEKVHSITGDRNSRLAVQLQDWDGNAESLQFSVHLGGEDTAYSLQLTEPVASQLGATTVPPSGLSVPFSTWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNLNGQYFRSIPQQRQELKKGI FWKTWRGRYYPLQATTMLIQPTAAEAASCyno ANGPTL4   4 ATGCGCGGTGCTCCGACGGCCGGAGCAGCCCTGATGCTCTGC(nucleic acid GTCGCCACGGCCGTGCTGCTGAGAGCTCAGGGCGGCCCGGTG sequence)CAGTCCAAGTCTCCGCGCTTTGCGTCCTGGGACGAGATGAATGTCCTGGCGCACGGACTCCTGCAGCTAGGCCAGGGGCTGCGCGAACACGCGGAGCGCACCCGCAGTCAGCTGAACGCGCTGGAGCGGCGCCTCAGCGCTTGCGGGTCTGCCTGCCAGGGAACCGAGGGGTCCACCGCCCTCCCGTTAGCCCCTGAGAGCCGGGTGGACCCTGAGGTCCTTCACAGCCTGCAGACACAACTCAAGGCTCAGAACAGCAGGATCCAGCAACTCTTCCACAAGGTGGCCCAGCAGCAGCGGCACCTGGAGAAGCAGCACCTGCGAATTCAGCGTCTGCAAAGCCAGGTTGGCCTCCTGGACCCCAAGCACCTAGACCATGAGGTGGCCAAGCCTGCCCGAAGAAAGAGGCGGCCCGAGATGGCCCAGCCAGTTGACTCGGCTCACAATGCCAGCCGCCTGCACCGGCTGCCCAGGGATTGCCAGGAGCTGTTTGAAGATGGGGAGAGGCAGAGTGGACTATTTGAGATCCAGCCTCAGGGGTCTCCGCCATTTTTGGTGAACTGCAAGATGACCTCAGATGGAGGCTGGACAGTAATTCAGAGGCGCCACGATGGCTCTGTGGACTTCAACCGGCCCTGGGAAGCCTACAAGGCGGGGTTTGGGGATCCCCAAGGCGAGTTCTGGCTGGGCCTGGAGAAGGTGCATAGCATCACAGGGGACCGCAACAGCCGCCTGGCCGTGCAGCTGCAGGACTGGGATGGCAACGCCGAGTCGCTGCAGTTCTCTGTGCACCTGGGTGGCGAGGACACGGCTTACAGCCTGCAGCTCACCGAGCCCGTGGCCAGCCAGTTGGGTGCCACCACCGTCCCGCCTAGCGGCCTCTCCGTACCCTTCTCCACTTGGGACCAGGATCACGACCTCCGCAGGGACAAGAACTGCGCCAAGAGCCTCTCTGGAGGCTGGTGGTTTGGCACCTGCAGCCATTCCAACCTCAATGGCCAGTACTTCCGCTCCATCCCACAGCAGCGGCAGGAGCTTAAGAAAGGAATCTTCTGGAAGACCTGGCGGGGCCGCTACTACCCGCTGCAGGCCACCACCATGTTGATCCAGCCCACGGCGGCAGAGGCAGCCTCC TAG Human ANGPTL3   5MFTIKLLLFIVPLVISSRIDQDNSSFDSLSPEPKSRFAMLDD amino acidVKILANGLLQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYD sequence (NCBILSLQTSEIKEEEKELRRTTYKLQVKNEEVKNMSLELNSKLES ReferenceLLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLKTFV NM_014495.3)EKQDNSIKDLLQTVEDQYKQLNQQHSQIKEIENQLRRTSIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFE Human ANGPTL3   6ATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTCTAGTT nucleic acidATTTCCTCCAGAATTGATCAAGACAATTCATCATTTGATTCT sequence (NCBICTATCTCCAGAGCCAAAATCAAGATTTGCTATGTTAGACGAT ReferenceGTAAAAATTTTAGCCAATGGCCTCCTTCAGTTGGGACATGGT NM_014495.3)CTTAAAGACTTTGTCCATAAGACGAAGGGCCAAATTAATGACATATTTCAAAAACTCAACATATTTGATCAGTCTTTTTATGATCTATCGCTGCAAACCAGTGAAATCAAAGAAGAAGAAAAGGAACTGAGAAGAACTACATATAAACTACAAGTCAAAAATGAAGAGGTAAAGAATATGTCACTTGAACTCAACTCAAAACTTGAAAGCCTCCTAGAAGAAAAAATTCTACTTCAACAAAAAGTGAAATATTTAGAAGAGCAACTAACTAACTTAATTCAAAATCAACCTGAAACTCCAGAACACCCAGAAGTAACTTCACTTAAAACTTTTGTAGAAAAACAAGATAATAGCATCAAAGACCTTCTCCAGACCGTGGAAGACCAATATAAACAATTAAACCAACAGCATAGTCAAATAAAAGAAATAGAAAATCAGCTCAGAAGGACTAGTATTCAAGAACCCACAGAAATTTCTCTATCTTCCAAGCCAAGAGCACCAAGAACTACTCCCTTTCTTCAGTTGAATGAAATAAGAAATGTAAAACATGATGGCATTCCTGCTGAATGTACCACCATTTATAACAGAGGTGAACATACAAGTGGCATGTATGCCATCAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTATATCAGGTAGTCCATGGACATTAATTCAACATCGAATAGATGGATCACAAAACTTCAATGAAACGTGGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTTGGTTGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAATTGAGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAAATCACGAAACCAACTATACGCTACATCTAGTTGCGATTACTGGCAATGTCCCCAATGCAATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCATGATGAGTGTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAGCAAAATCTAAGCCAGAGAGGAGAAGAGGATTATCTTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAA NEG276 HCDR1 (Kabat)   7 SSWMQHCDR2 (Kabat)   8 EIDPSDNYANYNQKFQG HCDR3 (Kabat)   9 GSYFSNFFDYHCDR1 (Chothia)  10 AYTFTSS HCDR2 (Chothia)  11 DPSDNY HCDR3 (Chothia) 12 GSYFSNFFDY VH  13 QVQLVQSGAEVKKPGASVKVSCKASAYTFTSSWMQWVRQAPGQGLEWMGEIDPSDNYANYNQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCASGSYFSNFFDYWGQGTLVTVSS DNA Encoding VH  14CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCAGGCGCCAGCGTGAAGGTGTCCTGCAAGGCCAGCGCCTACACCTTTACCAGCAGCTGGATGCAGTGGGTGCGCCAGGCTCCTGGACAGGGCCTGGAATGGATGGGCGAGATCGACCCCAGCGACAACTACGCCAACTACAACCAGAAATTCCAGGGCAGAGTGACCCTGACCGTGGACACCAGCACCTCCACCGCCTACATGGAACTGAGCAGCCTGCGGAGCGAGGACACCGCCGTGTACTATTGTGCCAGCGGCAGCTACTTCAGCAACTTCTTCGACTACTGGGGCCAGGGC ACCCTCGTGACCGTGTCATCTHeavy Chain  15 QVQLVQSGAEVKKPGASVKVSCKASAYTFTSSWMQWVRQAPGQGLEWMGEIDPSDNYANYNQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCASGSYFSNFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKDNA Encoding Heavy  16 CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCA ChainGGCGCCAGCGTGAAGGTGTCCTGCAAGGCCAGCGCCTACACCTTTACCAGCAGCTGGATGCAGTGGGTGCGCCAGGCTCCTGGACAGGGCCTGGAATGGATGGGCGAGATCGACCCCAGCGACAACTACGCCAACTACAACCAGAAATTCCAGGGCAGAGTGACCCTGACCGTGGACACCAGCACCTCCACCGCCTACATGGAACTGAGCAGCCTGCGGAGCGAGGACACCGCCGTGTACTATTGTGCCAGCGGCAGCTACTTCAGCAACTTCTTCGACTACTGGGGCCAGGGCACCCTCGTGACCGTGTCATCTGCTAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCCCCAGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGGC AAG LCDR1 (Kabat)  17KASQDIGSNLN LCDR2 (Kabat)  18 AVSNRGP LCDR3 (Kabat)  19 LQYASSPWTLCDR1 (Chothia)  20 SQDIGSN LCDR2 (Chothia)  21 AVS LCDR3 (Chothia)  32YASSPW VL  23 EIVMTQSPATLSVSPGERATLSCKASQDIGSNLNWLQQKPGQAPRRLIYAVSNRGPGIPARFSGSRSGSEYTLTISSLQSEDFA VYYCLQYASSPWTFGQGTKVEIKDNA Encoding VL  24 GAGATCGTGATGACACAGAGCCCCGCCACCCTGTCCGTGTCTCCAGGCGAAAGAGCCACCCTGAGCTGCAAAGCCAGCCAGGACATCGGCAGCAACCTGAACTGGCTGCAGCAGAAACCAGGCCAGGCCCCCAGAAGGCTGATCTACGCTGTTTCCAACCGTGGTCCTGGCATCCCCGCCAGATTTTCCGGCAGCAGATCCGGCAGCGAGTACACCCTGACCATCAGCAGCCTGCAGAGCGAGGACTTCGCCGTGTACTACTGCCTGCAGTACGCCAGCAGCCCCTGGACATTT GGCCAGGGCACCAAGGTGGAAATCAAGLight Chain  25 EIVMTQSPATLSVSPGERATLSCKASQDIGSNLNWLQQKPGQAPRRLIYAVSNRGPGIPARFSGSRSGSEYTLTISSLQSEDFAVYYCLQYASSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC DNA encoding Light  26GAGATCGTGATGACACAGAGCCCCGCCACCCTGTCCGTGTCT ChainCCAGGCGAAAGAGCCACCCTGAGCTGCAAAGCCAGCCAGGACATCGGCAGCAACCTGAACTGGCTGCAGCAGAAACCAGGCCAGGCCCCCAGAAGGCTGATCTACGCTGTTTCCAACCGTGGTCCTGGCATCCCCGCCAGATTTTCCGGCAGCAGATCCGGCAGCGAGTACACCCTGACCATCAGCAGCCTGCAGAGCGAGGACTTCGCCGTGTACTACTGCCTGCAGTACGCCAGCAGCCCCTGGACATTTGGCCAGGGCACCAAGGTGGAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAAC AGGGGCGAGTGC NEG276-LALAHCDR1 (Kabat)   7 SSWMQ HCDR2 (Kabat)   8 EIDPSDNYANYNQKFQGHCDR3 (Kabat)   9 GSYFSNFFDY HCDR1 (Chothia)  10 AYTFTSS HCDR2 (Chothia) 11 DPSDNY HCDR3 (Chothia)  12 GSYFSNFFDY VH  13QVQLVQSGAEVKKPGASVKVSCKASAYTFTSSWMQWVRQAPGQGLEWMGEIDPSDNYANYNQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCASGSYFSNFFDYWGQGTLVTVSS DNA Encoding VH  27CAGGTGCAGCTGGTGCAGTCAGGCGCCGAAGTGAAGAAACCCGGCGCTAGTGTGAAAGTCAGCTGTAAAGCTAGTGCCTACACCTTCACCTCTAGCTGGATGCAGTGGGTCAGACAGGCCCCAGGTCAGGGCCTGGAGTGGATGGGCGAGATCGACCCTAGCGATAACTACGCTAACTATAATCAGAAGTTTCAGGGTAGAGTCACCCTGACCGTGGACACTAGCACTAGCACCGCCTATATGGAACTGTCTAGCCTGAGATCAGAGGACACCGCCGTCTACTACTGCGCTAGTGGTAGCTACTTCTCTAACTTCTTCGACTACTGGGGTCAGGGC ACCCTGGTCACCGTGTCTAGCHeavy Chain  28 QVQLVQSGAEVKKPGASVKVSCKASAYTFTSSWMQWVRQAPGQGLEWMGEIDPSDNYANYNQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCASGSYFSNFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKDNA Encoding Heavy  29 CAGGTGCAGCTGGTGCAGTCAGGCGCCGAAGTGAAGAAACCC ChainGGCGCTAGTGTGAAAGTCAGCTGTAAAGCTAGTGCCTACACCTTCACCTCTAGCTGGATGCAGTGGGTCAGACAGGCCCCAGGTCAGGGCCTGGAGTGGATGGGCGAGATCGACCCTAGCGATAACTACGCTAACTATAATCAGAAGTTTCAGGGTAGAGTCACCCTGACCGTGGACACTAGCACTAGCACCGCCTATATGGAACTGTCTAGCCTGAGATCAGAGGACACCGCCGTCTACTACTGCGCTAGTGGTAGCTACTTCTCTAACTTCTTCGACTACTGGGGTCAGGGCACCCTGGTCACCGTGTCTAGCGCTAGCACTAAGGGCCCCTCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCCGGCGGCACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCTGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTGACCTCTGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTCACAGTGCCTTCAAGCAGCCTGGGCACCCAGACCTATATCTGCAACGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGCGGGTGGAGCCTAAGTCCTGCGACAAGACCCACACCTGTCCTCCCTGCCCTGCTCCTGAAGCTGCTGGCGGCCCTTCTGTGTTCCTGTTCCCTCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGATCCTGAAGTGAAGTTCAATTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAAGTCTCCAACAAGGCCCTGCCTGCCCCTATCGAAAAGACAATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTGTACACCCTGCCACCCAGCCGGGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAGTGGGAGTCTAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTCCCTGTCTCCCGGC AAG LCDR1 (Kabat)  17KASQDIGSNLN LCDR2 (Kabat)  18 AVSNRGP LCDR3 (Kabat)  19 LQYASSPWTLCDR1 (Chothia)  20 SQDIGSN LCDR2 (Chothia)  21 AVS LCDR3 (Chothia)  22YASSPW VL  23 EIVMTQSPATLSVSPGERATLSCKASQDIGSNLNWLQQKPGQAPRRLIYAVSNRGPGIPARFSGSRSGSEYTLTISSLQSEDFA VYYCLQYASSPWTFGQGTKVEIKDNA Encoding VL  30 GAGATCGTGATGACTCAGTCACCCGCTACCCTGAGCGTCAGCCCTGGCGAGCGGGCTACACTGAGCTGTAAAGCCTCTCAGGATATCGGCTCTAACCTGAACTGGCTGCAGCAGAAGCCCGGTCAGGCCCCTAGACGGCTGATCTACGCCGTGTCTAATAGAGGCCCCGGAATCCCCGCTAGGTTTAGCGGCTCTAGGTCAGGTTCAGAGTACACCCTGACTATCTCTAGCCTGCAGTCAGAGGACTTCGCCGTCTACTACTGCCTGCAGTACGCCTCTAGCCCCTGGACCTTC GGTCAGGGCACTAAGGTCGAGATTAAGLight Chain  25 EIVMTQSPATLSVSPGERATLSCKASQDIGSNLNWLQQKPGQAPRRLIYAVSNRGPGIPARFSGSRSGSEYTLTISSLQSEDFAVYYCLQYASSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC DNA Encoding Light  31GAGATCGTGATGACTCAGTCACCCGCTACCCTGAGCGTCAGC ChainCCTGGCGAGCGGGCTACACTGAGCTGTAAAGCCTCTCAGGATATCGGCTCTAACCTGAACTGGCTGCAGCAGAAGCCCGGTCAGGCCCCTAGACGGCTGATCTACGCCGTGTCTAATAGAGGCCCCGGAATCCCCGCTAGGTTTAGCGGCTCTAGGTCAGGTTCAGAGTACACCCTGACTATCTCTAGCCTGCAGTCAGAGGACTTCGCCGTCTACTACTGCCTGCAGTACGCCTCTAGCCCCTGGACCTTCGGTCAGGGCACTAAGGTCGAGATTAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAAC AGGGGCGAGTGC NEG278HCDR1 (Kabat)  32 SSWMQ HCDR2 (Kabat)  33 EIDPSDNYANYNQKFQGHCDR3 (Kabat)  34 GSYFSNFFDY HCDR1 (Chothia)  35 AYTFTSS HCDR2 (Chothia) 36 DPSDNY HCDR3 (Chothia)  37 GSYFSNFFDY VH  38QVQLVQSGAEVKKPGASVKVSCKASAYTFTSSWMQWVRQAPGQGLEWMGEIDPSDNYANYNQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCASGSYFSNFFDYWGQGTLVTVSS DNA Encoding VH  39CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCAGGCGCCAGCGTGAAGGTGTCCTGCAAGGCCAGCGCCTACACCTTTACCAGCAGCTGGATGCAGTGGGTGCGCCAGGCTCCTGGACAGGGCCTGGAATGGATGGGCGAGATCGACCCCAGCGACAACTACGCCAACTACAACCAGAAATTCCAGGGCAGAGTGACCCTGACCGTGGACACCAGCACCTCCACCGCCTACATGGAACTGAGCAGCCTGCGGAGCGAGGACACCGCCGTGTACTATTGTGCCAGCGGCAGCTACTTCAGCAACTTCTTCGACTACTGGGGCCAGGGC ACCCTCGTGACCGTGTCATCTHeavy Chain  40 QVQLVQSGAEVKKPGASVKVSCKASAYTFTSSWMQWVRQAPGQGLEWMGEIDPSDNYANYNQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCASGSYFSNFFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKDNA Encoding Heavy  41 CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCA ChainGGCGCCAGCGTGAAGGTGTCCTGCAAGGCCAGCGCCTACACCTTTACCAGCAGCTGGATGCAGTGGGTGCGCCAGGCTCCTGGACAGGGCCTGGAATGGATGGGCGAGATCGACCCCAGCGACAACTACGCCAACTACAACCAGAAATTCCAGGGCAGAGTGACCCTGACCGTGGACACCAGCACCTCCACCGCCTACATGGAACTGAGCAGCCTGCGGAGCGAGGACACCGCCGTGTACTATTGTGCCAGCGGCAGCTACTTCAGCAACTTCTTCGACTACTGGGGCCAGGGCACCCTCGTGACCGTGTCATCTGCTAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCCCCAGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGGC AAG LCDR1 (Kabat)  42KASQDIGSNLN LCDR2 (Kabat)  43 AASVREP LCDR3 (Kabat)  44 LQYASSPWTLCDR1 (Chothia)  45 SQDIGSN LCDR2 (Chothia)  46 AAS LCDR3 (Chothia)  47YASSPW VL  48 EIVMTQSPATLSVSPGERATLSCKASQDIGSNLNWLQQKPGQAPRRLIYAASVREPGIPARFSGSRSGSEYTLTISSLQSEDFA VYYCLQYASSPWTFGQGTKVEIKDNA Encoding VL  49 GAGATCGTGATGACACAGAGCCCCGCCACCCTGTCCGTGTCTCCAGGCGAAAGAGCCACCCTGAGCTGCAAAGCCAGCCAGGACATCGGCAGCAACCTGAACTGGCTGCAGCAGAAACCAGGCCAGGCCCCCAGAAGGCTGATCTACGCTGCTTCCGTCCGTGAGCCTGGCATCCCCGCCAGATTTTCCGGCAGCAGATCCGGCAGCGAGTACACCCTGACCATCAGCAGCCTGCAGAGCGAGGACTTCGCCGTGTACTACTGCCTGCAGTACGCCAGCAGCCCCTGGACATTT GGCCAGGGCACCAAGGTGGAAATCAAGLight Chain  50 EIVMTQSPATLSVSPGERATLSCKASQDIGSNLNWLQQKPGQAPRRLIYAASVREPGIPARFSGSRSGSEYTLTISSLQSEDFAVYYCLQYASSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC DNA Encoding Light  51GAGATCGTGATGACACAGAGCCCCGCCACCCTGTCCGTGTCT ChainCCAGGCGAAAGAGCCACCCTGAGCTGCAAAGCCAGCCAGGACATCGGCAGCAACCTGAACTGGCTGCAGCAGAAACCAGGCCAGGCCCCCAGAAGGCTGATCTACGCTGCTTCCGTCCGTGAGCCTGGCATCCCCGCCAGATTTTCCGGCAGCAGATCCGGCAGCGAGTACACCCTGACCATCAGCAGCCTGCAGAGCGAGGACTTCGCCGTGTACTACTGCCTGCAGTACGCCAGCAGCCCCTGGACATTTGGCCAGGGCACCAAGGTGGAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAAC AGGGGCGAGTGC NEG310HCDR1 (Kabat)  52 SYTMH HCDR2 (Kabat)  53 YINPSSGYTKYNQKFQGHCDR3 (Kabat)  54 GWLLLAMDY HCDR1 (Chothia)  55 GYTFTSY HCDR2 (Chothia) 56 NPSSGY HCDR3 (Chothia)  57 GWLLLAMDY VH  58QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYTMHWVRQAPGQGLEWMGYINPSSGYTKYNQKFQGRVTMTADKSTSTAYMELSSLRSEDTAVYYCAEGWLLLAMDYWGQGTLVTVSS DNA Encoding VH  59CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCAGGCGCCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTTACCAGCTACACCATGCACTGGGTGCGCCAGGCTCCAGGCCAGGGACTGGAATGGATGGGCTACATCAACCCCAGCAGCGGCTATACCAAGTACAACCAGAAATTCCAGGGCCGCGTGACCATGACCGCCGACAAGAGCACAAGCACCGCCTACATGGAACTGAGCAGCCTGCGGAGCGAGGACACCGCCGTGTACTATTGTGCCGAGGGCTGGCTGCTGCTGGCCATGGATTATTGGGGCCAGGGCACC CTCGTGACCGTGTCTAGTHeavy Chain  60 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYTMHWVRQAPGQGLEWMGYINPSSGYTKYNQKFQGRVTMTADKSTSTAYMELSSLRSEDTAVYYCAEGWLLLAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGKDNA Encoding Heavy  61 CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCA ChainGGCGCCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTTACCAGCTACACCATGCACTGGGTGCGCCAGGCTCCAGGCCAGGGACTGGAATGGATGGGCTACATCAACCCCAGCAGCGGCTATACCAAGTACAACCAGAAATTCCAGGGCCGCGTGACCATGACCGCCGACAAGAGCACAAGCACCGCCTACATGGAACTGAGCAGCCTGCGGAGCGAGGACACCGCCGTGTACTATTGTGCCGAGGGCTGGCTGCTGCTGGCCATGGATTATTGGGGCCAGGGCACCCTCGTGACCGTGTCTAGTGCTAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCCCCAGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGGCAAG LCDR1 (Kabat)  62RSSTGAVTTSNYAI LCDR2 (Kabat)  63 GTNNRAP LCDR3 (Kabat)  64 ALWYSDHWVLCDR1 (Chothia)  65 STGAVTTSNY LCDR2 (Chothia)  66 GTN LCDR3 (Chothia) 67 WYSDHW VL  68 EAVVTQSPATLSLSPGERATLSCRSSTGAVTTSNYAIWVQEKPGQAPRGLIGGTNNRAPGIPARFSGSLSGDDATLTISSLQPE DFAVYFCALWYSDHWVFGQGTKVEIKDNA Encoding VL  69 GAAGCCGTCGTGACACAGAGCCCTGCCACCCTGTCACTGAGCCCTGGCGAAAGAGCCACCCTGAGCTGCAGATCTAGCACCGGCGCTGTGACCACCAGCAACTACGCCATCTGGGTGCAGGAAAAGCCCGGCCAGGCTCCCAGAGGACTGATCGGCGGCACCAACAATAGAGCCCCTGGCATCCCCGCCAGATTCAGCGGATCTCTGTCTGGCGACGACGCCACACTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCGTGTACTTCTGCGCCCTGTGGTACAGCGACCACTGGGTGTTCGGCCAGGGCACCAAGGTGGAAATCAAG Light Chain  70EAVVTQSPATLSLSPGERATLSCRSSTGAVTTSNYAIWVQEKPGQAPRGLIGGTNNRAPGIPARFSGSLSGDDATLTISSLQPEDFAVYFCALWYSDHWVFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC DNA Encoding Light 71 GAAGCCGTCGTGACACAGAGCCCTGCCACCCTGTCACTGAGC ChainCCTGGCGAAAGAGCCACCCTGAGCTGCAGATCTAGCACCGGCGCTGTGACCACCAGCAACTACGCCATCTGGGTGCAGGAAAAGCCCGGCCAGGCTCCCAGAGGACTGATCGGCGGCACCAACAATAGAGCCCCTGGCATCCCCGCCAGATTCAGCGGATCTCTGTCTGGCGACGACGCCACACTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCGTGTACTTCTGCGCCCTGTGGTACAGCGACCACTGGGTGTTCGGCCAGGGCACCAAGGTGGAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAG AGCTTCAACAGGGGCGAGTGC NEG313HCDR1 (Kabat)  72 NYWIT HCDR2 (Kabat)  73 DFYPGGGSTNYNAKLQGHCDR3 (Kabat)  74 SPPQVAPFDY HCDR1 (Chothia)  75 GYTFNNY HCDR2 (Chothia) 76 YPGGGS HCDR3 (Chothia)  77 SPPQVAPFDY VH  78QVQLVQSGAEVKKPGASVKVSCKASGYTFNNYWITWVRQAPGQGLEWMGDFYPGGGSTNYNAKLQGRVTLTVDTSTSTAYMELRSLRSDDTAVYYCARSPPQVAPFDYWGQGTLVTVSS DNA encoding VH  79CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCAGGCGCCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTTAACAACTACTGGATCACCTGGGTGCGCCAGGCCCCTGGACAGGGACTGGAATGGATGGGCGACTTCTACCCTGGCGGCGGCAGCACCAACTACAACGCCAAGCTGCAGGGCAGAGTGACCCTGACCGTGGACACCAGCACCTCCACCGCCTACATGGAACTGCGGAGCCTGAGAAGCGACGACACCGCCGTGTATTACTGCGCTAGAAGCCCTCCTCAGGTGGCCCCCTTCGATTATTGGGGCCAGGGC ACACTCGTGACCGTGTCCTCTHeavy Chain  80 QVQLVQSGAEVKKPGASVKVSCKASGYTFNNYWITWVRQAPGQGLEWMGDFYPGGGSTNYNAKLQGRVTLTVDTSTSTAYMELRSLRSDDTAVYYCARSPPQVAPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKDNA Encoding Heavy  81 CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCA ChainGGCGCCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTTAACAACTACTGGATCACCTGGGTGCGCCAGGCCCCTGGACAGGGACTGGAATGGATGGGCGACTTCTACCCTGGCGGCGGCAGCACCAACTACAACGCCAAGCTGCAGGGCAGAGTGACCCTGACCGTGGACACCAGCACCTCCACCGCCTACATGGAACTGCGGAGCCTGAGAAGCGACGACACCGCCGTGTATTACTGCGCTAGAAGCCCTCCTCAGGTGGCCCCCTTCGATTATTGGGGCCAGGGCACACTCGTGACCGTGTCCTCTGCTAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCCCCAGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGGC AAG LCDR1 (Kabat)  82QASDYIYHWLG LCDR2 (Kabat)  83 GASGLET LCDR3 (Kabat)  84 QQYWSTPWTLCDR1 (Chothia)  85 SDYIYHW LCDR2 (Chothia)  86 GAS LCDR3 (Chothia)  87YWSTPW VL  88 DIQMTQSPSSLSASVGDRVTITCQASDYIYHWLGWYQQKPGKAPKLLISGASGLETGVPSRFSGSGSGKDYTFTISSLQPEDIA TYYCQQYWSTPWTFGQGTKLEIKDNA Encoding VL  89 GACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCCAGCGTGGGCGACAGGGTGACCATCACCTGTCAGGCCAGCGACTACATCTACCACTGGCTGGGCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATTAGCGGAGCCTCCGGTCTGGAAACCGGCGTGCCAAGCAGATTTTCCGGCAGCGGCTCCGGCAAGGACTACACCTTCACCATCAGCTCCCTGCAGCCCGAGGATATCGCCACCTACTACTGCCAGCAGTACTGGTCCACCCCCTGGACCTTT GGCCAGGGCACCAAGCTGGAAATCAAGLight Chain  90 DIQMTQSPSSLSASVGDRVTITCQASDYIYHWLGWYQQKPGKAPKLLISGASGLETGVPSRFSGSGSGKDYTFTISSLQPEDIATYYCQQYWSTPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC DNA Encoding Light  91GACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCCAGC ChainGTGGGCGACAGGGTGACCATCACCTGTCAGGCCAGCGACTACATCTACCACTGGCTGGGCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATTAGCGGAGCCTCCGGTCTGGAAACCGGCGTGCCAAGCAGATTTTCCGGCAGCGGCTCCGGCAAGGACTACACCTTCACCATCAGCTCCCTGCAGCCCGAGGATATCGCCACCTACTACTGCCAGCAGTACTGGTCCACCCCCTGGACCTTTGGCCAGGGCACCAAGCTGGAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAAC AGGGGCGAGTGC NEG315HCDR1 (Kabat)  92 NYWIT HCDR2 (Kabat)  93 DFYPGGGNTNYNAKLQGHCDR3 (Kabat)  94 SPPQVAPFDY HCDR1 (Chothia)  95 GYTFTNY HCDR2 (Chothia) 96 YPGGGN HCDR3 (Chothia)  97 SPPQVAPFDY VH  98QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWITWVRQAPGQGLEWMGDFYPGGGNTNYNAKLQGRVTLTVDTSTSTAYMELRSLRSDDTAVYYCARSPPQVAPFDYWGQGTLVTVSS DNA Encoding VH  99CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCAGGCGCCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTTACCAACTACTGGATCACCTGGGTGCGCCAGGCCCCTGGACAGGGACTGGAATGGATGGGCGACTTCTACCCTGGCGGCGGCAACACCAACTACAACGCCAAGCTGCAGGGCAGAGTGACCCTGACCGTGGACACCAGCACCTCCACCGCCTACATGGAACTGCGGAGCCTGAGAAGCGACGACACCGCCGTGTATTACTGCGCTAGAAGCCCTCCTCAGGTGGCCCCCTTCGATTATTGGGGCCAGGGC ACACTCGTGACCGTGTCCTCTHeavy Chain 100 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWITWVRQAPGQGLEWMGDFYPGGGNTNYNAKLQGRVTLTVDTSTSTAYMELRSLRSDDTAVYYCARSPPQVAPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKDNA Encoding Heavy 101 CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCA ChainGGCGCCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTTACCAACTACTGGATCACCTGGGTGCGCCAGGCCCCTGGACAGGGACTGGAATGGATGGGCGACTTCTACCCTGGCGGCGGCAACACCAACTACAACGCCAAGCTGCAGGGCAGAGTGACCCTGACCGTGGACACCAGCACCTCCACCGCCTACATGGAACTGCGGAGCCTGAGAAGCGACGACACCGCCGTGTATTACTGCGCTAGAAGCCCTCCTCAGGTGGCCCCCTTCGATTATTGGGGCCAGGGCACACTCGTGACCGTGTCCTCTGCTAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCCCCAGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGGC AAG LCDR1 (Kabat) 102QASEYIYNWLG LCDR2 (Kabat) 103 GASGLET LCDR3 (Kabat) 104 QQYWSTPWTLCDR1 (Chothia) 105 SEYIYNW LCDR2 (Chothia) 106 GAS LCDR3 (Chothia) 107YWSTPW VL 108 DIQMTQSPSSLSASVGDRVTITCQASEYIYNWLGWYQQKPGKAPKLLISGASGLETGVPSRFSGSGSGKDYTFTISSLQPEDIA TYYCQQYWSTPWTFGQGTKLEIKDNA Encoding VL 109 GACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCCAGCGTGGGCGACAGGGTGACCATCACCTGTCAGGCCAGCGAATACATCTACAACTGGCTGGGCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATTAGCGGAGCCTCCGGTCTGGAAACCGGCGTGCCAAGCAGATTTTCCGGCAGCGGCTCCGGCAAGGACTACACCTTCACCATCAGCTCCCTGCAGCCCGAGGATATCGCCACCTACTACTGCCAGCAGTACTGGTCCACCCCCTGGACCTTT GGCCAGGGCACCAAGCTGGAAATCAAGLight Chain 110 DIQMTQSPSSLSASVGDRVTITCQASEYIYNWLGWYQQKPGKAPKLLISGASGLETGVPSRFSGSGSGKDYTFTISSLQPEDIATYYCQQYWSTPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC DNA Encoding Light 111GACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCCAGC ChainGTGGGCGACAGGGTGACCATCACCTGTCAGGCCAGCGAATACATCTACAACTGGCTGGGCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATTAGCGGAGCCTCCGGTCTGGAAACCGGCGTGCCAAGCAGATTTTCCGGCAGCGGCTCCGGCAAGGACTACACCTTCACCATCAGCTCCCTGCAGCCCGAGGATATCGCCACCTACTACTGCCAGCAGTACTGGTCCACCCCCTGGACCTTTGGCCAGGGCACCAAGCTGGAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAAC AGGGGCGAGTGC NEG318HCDR1 (Kabat) 112 SFWIT HCDR2 (Kabat) 113 DIYPGGATTNYNEKLQGHCDR3 (Kabat) 114 SPPQVGPFDY HCDR1 (Chothia) 115 GYTFTSF HCDR2 (Chothia)116 YPGGAT HCDR3 (Chothia) 117 SPPQVGPFDY VH 118QVQLVQSGAEVKKPGASVKVSCKASGYTFTSFWITWVRQAPGQGLEWMGDIYPGGATTNYNEKLQGRVTLTVDTSTSTAYMELRSLRSDDTAVYYCARSPPQVGPFDYWGQGTLVTVSS DNA Encoding VH 119CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCAGGCGCCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTATACCTTCACCAGCTTTTGGATCACCTGGGTGCGCCAGGCCCCTGGACAGGGACTGGAATGGATGGGCGACATCTACCCTGGCGGCGCCACCACCAACTACAACGAGAAGCTGCAGGGCAGAGTGACCCTGACCGTGGACACCAGCACCTCCACCGCCTACATGGAACTGCGGAGCCTGAGAAGCGACGACACCGCCGTGTACTACTGCGCTAGAAGCCCTCCTCAGGTGGGCCCCTTCGATTATTGGGGCCAGGGC ACACTCGTGACCGTGTCCTCTHeavy Chain 120 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSFWITWVRQAPGQGLEWMGDIYPGGATTNYNEKLQGRVTLTVDTSTSTAYMELRSLRSDDTAVYYCARSPPQVGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKDNA Encoding Heavy 121 CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCA ChainGGCGCCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTATACCTTCACCAGCTTTTGGATCACCTGGGTGCGCCAGGCCCCTGGACAGGGACTGGAATGGATGGGCGACATCTACCCTGGCGGCGCCACCACCAACTACAACGAGAAGCTGCAGGGCAGAGTGACCCTGACCGTGGACACCAGCACCTCCACCGCCTACATGGAACTGCGGAGCCTGAGAAGCGACGACACCGCCGTGTACTACTGCGCTAGAAGCCCTCCTCAGGTGGGCCCCTTCGATTATTGGGGCCAGGGCACACTCGTGACCGTGTCCTCTGCTAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCCCCAGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGGC AAG LCDR1 (Kabat) 122QASDYIYHWLA LCDR2 (Kabat) 123 GASSLET LCDR3 (Kabat) 124 QQYWSIPWTLCDR1 (Chothia) 125 SDYIYHW LCDR2 (Chothia) 126 GAS LCDR3 (Chothia) 127YWSIPW VL 128 DIQMTQSPSSLSASVGDRVTITCQASDYIYHWLAWYQQKPGKAPKLLISGASSLETGVPSRFSGSGSGKDYTFTISSLQPEDIA TYYCQQYWSIPWTFGQGTKLEIKDNA Encoding VL 129 GACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCACCTGTCAGGCCAGCGACTACATCTACCACTGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATTAGCGGAGCCTCCAGTCTGGAAACCGGCGTGCCAAGCAGATTTTCCGGCAGCGGCTCCGGCAAGGACTACACCTTCACCATCAGCTCCCTGCAGCCCGAGGATATCGCCACCTACTACTGCCAGCAGTACTGGTCCATCCCCTGGACCTTT GGCCAGGGCACCAAGCTGGAAATCAAGLight Chain 130 DIQMTQSPSSLSASVGDRVTITCQASDYIYHWLAWYQQKPGKAPKLLISGASSLETGVPSRFSGSGSGKDYTFTISSLQPEDIATYYCQQYWSIPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC DNA Encoding Light 131GACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCCAGC ChainGTGGGCGACAGAGTGACCATCACCTGTCAGGCCAGCGACTACATCTACCACTGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATTAGCGGAGCCTCCAGTCTGGAAACCGGCGTGCCAAGCAGATTTTCCGGCAGCGGCTCCGGCAAGGACTACACCTTCACCATCAGCTCCCTGCAGCCCGAGGATATCGCCACCTACTACTGCCAGCAGTACTGGTCCATCCCCTGGACCTTTGGCCAGGGCACCAAGCTGGAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAAC AGGGGCGAGTGC NEG319HCDR1 (Kabat) 132 SFWIT HCDR2 (Kabat) 133 DIYPGGANTNYNEKLQGHCDR3 (Kabat) 134 SPPQVGPFDY HCDR1 (Chothia) 135 GYTFTSF HCDR2 (Chothia)136 YPGGAN HCDR3 (Chothia) 137 SPPQVGPFDY VH 138QVQLVQSGAEVKKPGASVKVSCKASGYTFTSFWITWVRQAPGQGLEWMGDIYPGGANTNYNEKLQGRVTLTVDTSTSTAYMELRSLRSDDTAVYYCARSPPQVGPFDYWGQGTLVTVSS DNA Encoding VH 139CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCAGGCGCCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTATACCTTCACCAGCTTTTGGATCACCTGGGTGCGCCAGGCCCCTGGACAGGGACTGGAATGGATGGGCGACATCTACCCTGGCGGCGCCAACACCAACTACAACGAGAAGCTGCAGGGCAGAGTGACCCTGACCGTGGACACCAGCACCTCCACCGCCTACATGGAACTGCGGAGCCTGAGAAGCGACGACACCGCCGTGTACTACTGCGCTAGAAGCCCTCCTCAGGTGGGCCCCTTCGATTATTGGGGCCAGGGC ACACTCGTGACCGTGTCCTCTHeavy Chain 140 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSFWITWVRQAPGQGLEWMGDIYPGGANTNYNEKLQGRVTLTVDTSTSTAYMELRSLRSDDTAVYYCARSPPQVGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKDNA Encoding Heavy 141 CAGGTGCAGCTGGTGCAGTCTGGCGCCGAAGTGAAGAAACCA ChainGGCGCCAGCGTGAAGGTGTCCTGCAAGGCCAGCGGCTATACCTTCACCAGCTTTTGGATCACCTGGGTGCGCCAGGCCCCTGGACAGGGACTGGAATGGATGGGCGACATCTACCCTGGCGGCGCCAACACCAACTACAACGAGAAGCTGCAGGGCAGAGTGACCCTGACCGTGGACACCAGCACCTCCACCGCCTACATGGAACTGCGGAGCCTGAGAAGCGACGACACCGCCGTGTACTACTGCGCTAGAAGCCCTCCTCAGGTGGGCCCCTTCGATTATTGGGGCCAGGGCACACTCGTGACCGTGTCCTCTGCTAGCACCAAGGGCCCCAGCGTGTTCCCCCTGGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGTCCAGCGTGGTGACAGTGCCCAGCAGCAGCCTGGGCACCCAGACCTACATCTGCAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTGGAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCAGCCCCAGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCACGAGGACCCAGAGGTGAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAATACAAGTGCAAGGTCTCCAACAAGGCCCTGCCAGCCCCCATCGAAAAGACCATCAGCAAGGCCAAGGGCCAGCCACGGGAGCCCCAGGTGTACACCCTGCCCCCCTCCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCAGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAAGTCCAGGTGGCAGCAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGTCCCCCGGC AAG LCDR1 (Kabat) 142QASEYIINWLA LCDR2 (Kabat) 143 GATGLET LCDR3 (Kabat) 144 QQYWSIPWTLCDR1 (Chothia) 145 SEYIINW LCDR2 (Chothia) 146 GAT LCDR3 (Chothia) 147YWSIPW VL 148 DIQMTQSPSSLSASVGDRVTITCQASEYIINWLAWYQQKPGKAPKLLISGATGLETGVPSRFSGSGSGKDYTFTISSLQPEDIA TYYCQQYWSIPWTFGQGTKLEIKDNA Encoding VL 149 GACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCACCTGTCAGGCCAGCGAATACATCATAAACTGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATTAGCGGAGCCACCGGTCTGGAAACCGGCGTGCCAAGCAGATTTTCCGGCAGCGGCTCCGGCAAGGACTACACCTTCACCATCAGCTCCCTGCAGCCCGAGGATATCGCCACCTACTACTGCCAGCAGTACTGGTCCATCCCCTGGACCTTT GGCCAGGGCACCAAGCTGGAAATCAAGLight Chain 150 DIQMTQSPSSLSASVGDRVTITCQASEYIINWLAWYQQKPGKAPKLLISGATGLETGVPSRFSGSGSGKDYTFTISSLQPEDIATYYCQQYWSIPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC DNA Encoding Light 151GACATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCCAGC ChainGTGGGCGACAGAGTGACCATCACCTGTCAGGCCAGCGAATACATCATAAACTGGCTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATTAGCGGAGCCACCGGTCTGGAAACCGGCGTGCCAAGCAGATTTTCCGGCAGCGGCTCCGGCAAGGACTACACCTTCACCATCAGCTCCCTGCAGCCCGAGGATATCGCCACCTACTACTGCCAGCAGTACTGGTCCATCCCCTGGACCTTTGGCCAGGGCACCAAGCTGGAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAAC AGGGGCGAGTGC

Other antibodies of the invention include those where the amino acids ornucleic acids encoding the amino acids have been mutated, yet have atleast 60, 65, 70, 75, 80, 85, 90, or 95 percent identity to thesequences described in Table 1. Some embodiments include mutant aminoacid sequences wherein no more than 1, 2, 3, 4 or 5 amino acids havebeen mutated in the variable regions when compared with the variableregions depicted in the sequence described in Table 1, while retainingsubstantially the same antigen binding activity.

Since each of these antibodies can bind to ANGPTL4, the VH, VL,full-length light chain, and full-length heavy chain sequences (aminoacid sequences and the nucleotide sequences encoding the amino acidsequences) can be “mixed and matched” to create other ANGPTL4-bindingantibodies of the invention. Such “mixed and matched” ANGPTL4-bindingantibodies can be tested using the binding assays known in the art(e.g., ELISAs, and other assays described in the Example section). Whenthese chains are mixed and matched, a VH sequence from a particularVH/VL pairing should be replaced with a structurally similar VHsequence. Likewise a full-length heavy chain sequence from a particularfull-length heavy chain/full length light chain pairing should bereplaced with a structurally similar full-length heavy chain sequence.Likewise, a VL sequence from a particular VH/VL pairing should bereplaced with a structurally similar VL sequence. Likewise a full-lengthlight chain sequence from a particular full-length heavychain/full-length light chain pairing should be replaced with astructurally similar full-length light chain sequence.

Accordingly, in one aspect, the invention provides an isolated antibodyor antigen binding region thereof having: a heavy chain variable domaincomprising an amino acid sequence selected from the group consisting ofSEQ ID NOs: 13, 38, 58, 78, 98, 118, and 138, and a light chain variabledomain comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 23, 48, 68, 88, 108, 128, and 148, wherein theantibody specifically binds to ANGPTL4 (e.g., human ANGPTL4).

More specifically, in certain aspects, the invention provides anisolated antibody or antigen binding region thereof having a heavy chainvariable domain and a light chain variable domain comprising amino acidsequences selected from SEQ ID NOs: 13 and 23; 38 and 48; 58 and 68; 78and 88; 98 and 108, 118 and 128, or 138 and 148, respectively.

In another aspect, the invention provides (i) an isolated antibodyhaving: a full-length heavy chain comprising an amino acid sequence thathas been optimized for expression in a mammalian cell selected from thegroup consisting of SEQ ID NOs: 15, 28, 40, 60, 80, 100, 120, and 140,and a full-length light chain comprising an amino acid sequence that hasbeen optimized for expression in a mammalian cell selected from thegroup consisting of SEQ ID NOs: 25, 50, 70, 90, 110, 130, and 150; or(ii) a functional protein comprising an antigen binding portion thereof.More specifically, in certain aspects, the invention provides anisolated antibody or antigen binding region thereof having a heavy chainand a light chain comprising amino acid sequences selected from SEQ IDNOs: 15 and 25; 28 and 25; 40 and 50; 60 and 70; 80 and 90; 100 and 110;120 and 130; or 140 and 150, respectively.

The terms “complementarity determining region,” and “CDR,” as usedherein refer to the sequences of amino acids within antibody variableregions which confer antigen specificity and binding affinity. Ingeneral, there are three CDRs in each heavy chain variable region(HCDR1, HCDR2, HCDR3) and three CDRs in each light chain variable region(LCDR1, LCDR2, LCDR3).

The precise amino acid sequence boundaries of a given CDR can be readilydetermined using any of a number of well-known schemes, including thosedescribed by Kabat et al. (1991), “Sequences of Proteins ofImmunological Interest,” 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (“Kabat” numbering scheme),Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numberingscheme).

For example, under Kabat, the CDR amino acid residues of antibody FF1 inthe heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-66(HCDR2), and 99-104 (HCDR3); and the CDR amino acid residues in thelight chain variable domain (VL) are numbered 24-34 (LCDR1), 50-55(LCDR2), and 89-97 (LCDR3). Under Chothia the CDR amino acids in the VHare numbered 26-32 (HCDR1), 52-57 (HCDR2), and 99-104 (HCDR3); and theamino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and91-96 (LCDR3). By combining the CDR definitions of both Kabat andChothia, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-66(HCDR2), and 90-104 (HCDR3) in human VH and amino acid residues 24-34(LCDR1), 50-55 (LCDR2), and 89-97 (LCDR3) in human VL.

In another aspect, the present invention provides ANGPTL4 bindingantibodies that comprise the heavy chain and light chain CDR1s, CDR2s,and CDR3s as described in Table 1, or combinations thereof. The aminoacid sequences of the VH CDR1s of the antibodies are shown in SEQ IDNOs: 7, 32, 52, 72, 92, 112, and 132. The amino acid sequences of the VHCDR2s of the antibodies and are shown in SEQ ID NOs: 8, 33, 53, 73, 93,113, and 133. The amino acid sequences of the VH CDR3s of the antibodiesare shown in SEQ ID NOs: 9, 34, 54, 74, 94, 114, and 134. The amino acidsequences of the VL CDR1s of the antibodies are shown in SEQ ID NOs: 17,42, 62, 82, 102, 122, and 142. The amino acid sequences of the VL CDR2sof the antibodies are shown in SEQ ID NOs: 18, 43, 63, 83, 103, 123, and143. The amino acid sequences of the VL CDR3s of the antibodies areshown in SEQ ID NOs: 19, 44, 64, 84, 104, 124, and 144. These CDRregions are delineated using the Kabat system.

Alternatively, as defined using the Chothia system (Al-Lazikani et al.,(1997) JMB 273, 927-948), the amino acid sequences of the VH CDR1s ofthe antibodies are shown in SEQ ID NOs: 10, 35, 55, 75, 95, 115, and135. The amino acid sequences of the VH CDR2s of the antibodies and areshown in SEQ ID NOs: 11, 36, 56, 76, 96, 116, and 136. The amino acidsequences of the VH CDR3s of the antibodies are shown in SEQ ID NOs: 12,37, 57, 77, 97, 117, 117, and 137. The amino acid sequences of the VLCDR1s of the antibodies are shown in SEQ ID NOs: 20, 45, 65, 85, 105,125, and 145. The amino acid sequences of the VL CDR2s of the antibodiesare shown in SEQ ID NOs: 21, 46, 66, 86, 106, 126, and 146. The aminoacid sequences of the VL CDR3s of the antibodies are shown in SEQ IDNOs: 22, 47, 67, 87, 107, 127, and 147.

Given that each of these antibodies can bind to ANGPTL4 and thatantigen-binding specificity is provided primarily by the CDR1, 2 and 3regions, the VH CDR1, 2 and 3 sequences and VL CDR1, 2 and 3 sequencescan be “mixed and matched” (i.e., CDRs from different antibodies can bemixed and matched, although each antibody preferably contains a VH CDR1,2 and 3 and a VL CDR1, 2 and 3 to create other ANGPTL4 binding moleculesof the invention. Such “mixed and matched” ANGPTL4 binding antibodiescan be tested using the binding assays known in the art and thosedescribed in the Examples (e.g., ELISAs, SET, Biacore). When VH CDRsequences are mixed and matched, the CDR1, CDR2 and/or CDR3 sequencefrom a particular VH sequence should be replaced with a structurallysimilar CDR sequence(s). Likewise, when VL CDR sequences are mixed andmatched, the CDR1, CDR2 and/or CDR3 sequence from a particular VLsequence should be replaced with a structurally similar CDR sequence(s).It will be readily apparent to the ordinarily skilled artisan that novelVH and VL sequences can be created by substituting one or more VH and/orVL CDR region sequences with structurally similar sequences from the CDRsequences shown herein for monoclonal antibodies of the presentinvention. In addition to the foregoing, in one embodiment, the antigenbinding fragments of the antibodies described herein can comprise a VHCDR1, 2, and 3, or a VL CDR 1, 2, and 3, wherein the fragment binds toANGPTL4 as a single variable domain.

In certain embodiments of the invention, the antibodies or antigenbinding fragments thereof may have the heavy and light chain sequencesof the Humanized antibodies described in Table 1. More specifically, theantibody or antigen binding fragments thereof may have the heavy andlight sequence of NEG276, NEG276-LALA, NEG278, NEG310, NEG313, NEG315,NEG318, and NEG319.

In other embodiments of the invention the antibody or antigen bindingfragment in that specifically binds ANGPTL4 comprises a heavy chainvariable region CDR1, a heavy chain variable region CDR2, a heavy chainvariable region CDR3, a light chain variable region CDR1, a light chainvariable region CDR2, and a light chain variable region CDR3 as definedby Kabat and described in Table 1. In still other embodiments of theinvention the antibody or antigen binding fragment in that specificallybinds ANGPTL4 comprises a heavy chain variable region CDR1, a heavychain variable region CDR2, a heavy chain variable region CDR3, a lightchain variable region CDR1, a light chain variable region CDR2, and alight chain variable region CDR3 as defined by Chothia and described inTable 1.

In a specific embodiment, the invention includes an antibody thatspecifically binds to ANGPTL4 comprising a heavy chain variable regionCDR1 of SEQ ID NO: 7; a heavy chain variable region CDR2 of SEQ ID NO:8; a heavy chain variable region CDR3 of SEQ ID NO: 9; a light chainvariable region CDR1 of SEQ ID NO: 17; a light chain variable regionCDR2 of SEQ ID NO: 18; and a light chain variable region CDR3 of SEQ IDNO: 19.

In another specific embodiment, the invention includes an antibody thatspecifically binds to ANGPTL4 comprising a heavy chain variable regionCDR1 of SEQ ID NO: 32; a heavy chain variable region CDR2 of SEQ ID NO:33; a heavy chain variable region CDR3 of SEQ ID NO: 34; a light chainvariable region CDR1 of SEQ ID NO: 42; a light chain variable regionCDR2 of SEQ ID NO: 43; and a light chain variable region CDR3 of SEQ IDNO: 44.

In another specific embodiment, the invention includes an antibody thatspecifically binds to ANGPTL4 comprising a heavy chain variable regionCDR1 of SEQ ID NO: 52; a heavy chain variable region CDR2 of SEQ ID NO:53; a heavy chain variable region CDR3 of SEQ ID NO: 54; a light chainvariable region CDR1 of SEQ ID NO: 62; a light chain variable regionCDR2 of SEQ ID NO: 63; and a light chain variable region CDR3 of SEQ IDNO: 64.

In another specific embodiment, the invention includes an antibody thatspecifically binds to ANGPTL4 comprising a heavy chain variable regionCDR1 of SEQ ID NO: 72; a heavy chain variable region CDR2 of SEQ ID NO:73; a heavy chain variable region CDR3 of SEQ ID NO: 74; a light chainvariable region CDR1 of SEQ ID NO: 82; a light chain variable regionCDR2 of SEQ ID NO: 83; and a light chain variable region CDR3 of SEQ IDNO: 84.

In another specific embodiment, the invention includes an antibody thatspecifically binds to ANGPTL4 comprising a heavy chain variable regionCDR1 of SEQ ID NO: 92; a heavy chain variable region CDR2 of SEQ ID NO:93; a heavy chain variable region CDR3 of SEQ ID NO: 94; a light chainvariable region CDR1 of SEQ ID NO: 102; a light chain variable regionCDR2 of SEQ ID NO: 103; and a light chain variable region CDR3 of SEQ IDNO: 104.

In another specific embodiment, the invention includes an antibody thatspecifically binds to ANGPTL4 comprising a heavy chain variable regionCDR1 of SEQ ID NO: 112; a heavy chain variable region CDR2 of SEQ ID NO:113; a heavy chain variable region CDR3 of SEQ ID NO: 114; a light chainvariable region CDR1 of SEQ ID NO: 122; a light chain variable regionCDR2 of SEQ ID NO: 123; and a light chain variable region CDR3 of SEQ IDNO: 124.

In another specific embodiment, the invention includes an antibody thatspecifically binds to ANGPTL4 comprising a heavy chain variable regionCDR1 of SEQ ID NO: 132; a heavy chain variable region CDR2 of SEQ ID NO:133; a heavy chain variable region CDR3 of SEQ ID NO: 134; a light chainvariable region CDR1 of SEQ ID NO: 142; a light chain variable regionCDR2 of SEQ ID NO: 143; and a light chain variable region CDR3 of SEQ IDNO: 144.

In another specific embodiment, the invention includes an antibody thatspecifically binds to ANGPTL4 comprising a heavy chain variable regionCDR1 of SEQ ID NO: 10; a heavy chain variable region CDR2 of SEQ ID NO:11; a heavy chain variable region CDR3 of SEQ ID NO: 12; a light chainvariable region CDR1 of SEQ ID NO: 20; a light chain variable regionCDR2 of SEQ ID NO: 21; and a light chain variable region CDR3 of SEQ IDNO: 22.

In another specific embodiment, the invention includes an antibody thatspecifically binds to ANGPTL4 comprising a heavy chain variable regionCDR1 of SEQ ID NO: 35; a heavy chain variable region CDR2 of SEQ ID NO:36; a heavy chain variable region CDR3 of SEQ ID NO: 37; a light chainvariable region CDR1 of SEQ ID NO: 45; a light chain variable regionCDR2 of SEQ ID NO: 46; and a light chain variable region CDR3 of SEQ IDNO: 47.

In another specific embodiment, the invention includes an antibody thatspecifically binds to ANGPTL4 comprising a heavy chain variable regionCDR1 of SEQ ID NO: 55; a heavy chain variable region CDR2 of SEQ ID NO:56; a heavy chain variable region CDR3 of SEQ ID NO: 57; a light chainvariable region CDR1 of SEQ ID NO: 65; a light chain variable regionCDR2 of SEQ ID NO: 66; and a light chain variable region CDR3 of SEQ IDNO: 67.

In another specific embodiment, the invention includes an antibody thatspecifically binds to ANGPTL4 comprising a heavy chain variable regionCDR1 of SEQ ID NO: 75; a heavy chain variable region CDR2 of SEQ ID NO:76; a heavy chain variable region CDR3 of SEQ ID NO: 77; a light chainvariable region CDR1 of SEQ ID NO: 85; a light chain variable regionCDR2 of SEQ ID NO: 86; and a light chain variable region CDR3 of SEQ IDNO: 87.

In another specific embodiment, the invention includes an antibody thatspecifically binds to ANGPTL4 comprising a heavy chain variable regionCDR1 of SEQ ID NO: 95; a heavy chain variable region CDR2 of SEQ ID NO:96; a heavy chain variable region CDR3 of SEQ ID NO: 97; a light chainvariable region CDR1 of SEQ ID NO: 105; a light chain variable regionCDR2 of SEQ ID NO: 106; and a light chain variable region CDR3 of SEQ IDNO: 107.

In another specific embodiment, the invention includes an antibody thatspecifically binds to ANGPTL4 comprising a heavy chain variable regionCDR1 of SEQ ID NO: 115; a heavy chain variable region CDR2 of SEQ ID NO:116; a heavy chain variable region CDR3 of SEQ ID NO: 117; a light chainvariable region CDR1 of SEQ ID NO: 125; a light chain variable regionCDR2 of SEQ ID NO: 126; and a light chain variable region CDR3 of SEQ IDNO: 127.

In another specific embodiment, the invention includes an antibody thatspecifically binds to ANGPTL4 comprising a heavy chain variable regionCDR1 of SEQ ID NO: 135; a heavy chain variable region CDR2 of SEQ ID NO:136; a heavy chain variable region CDR3 of SEQ ID NO: 137; a light chainvariable region CDR1 of SEQ ID NO: 145; a light chain variable regionCDR2 of SEQ ID NO: 146; and a light chain variable region CDR3 of SEQ IDNO: 147.

In certain embodiments, the invention includes antibodies or antigenbinding fragments that specifically bind to ANGPTL4 as described inTable 1. In a preferred embodiment, the antibody, or antigen bindingfragment, that binds ANGPTL4 is NEG276, NEG276-LALA, NEG278, NEG310,NEG313, NEG315, NEG318, NEG319.

Homologous Antibodies

In yet another embodiment, the present invention provides an antibody,or an antigen binding fragment thereof, comprising amino acid sequencesthat are homologous to the sequences described in Table 1, and theantibody binds to a ANGPTL4 protein (e.g., human and cynomolgus monkeyANGPTL4), and retains the desired functional properties of thoseantibodies described in Table 1.

For example, the invention provides an isolated antibody, or afunctional antigen binding fragment thereof, comprising a heavy chainvariable domain and a light chain variable domain, wherein the heavychain variable domain comprises an amino acid sequence that is at least80%, at least 90%, or at least 95% identical to an amino acid sequenceselected from the group consisting of SEQ ID NOs: 13, 38, 58, 78, 98,118, and 138; the light chain variable domain comprises an amino acidsequence that is at least 80%, at least 90%, or at least 95% identicalto an amino acid sequence selected from the group consisting of SEQ IDNOs: 23, 23, 48, 68, 88, 108, 128, 148; and the antibody specificallybinds to ANGPTL4 (e.g., human and cynomolgus monkey ANGPTL4). In certainaspects of the invention the heavy and light chain sequences furthercomprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences asdefined by Kabat, for example SEQ ID NOs: 7, 8, 9, 17, 18, and 19,respectively. In certain other aspects of the invention the heavy andlight chain sequences further comprise HCDR1, HCDR2, HCDR3, LCDR1,LCDR2, and LCDR3 sequences as defined by Chothia, for example SEQ IDNOs: 10, 11, 12, 20, 21, and 22, respectively.

In other embodiments, the VH and/or VL amino acid sequences may be 50%,60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequencesset forth in Table 1. In other embodiments, the VH and/or VL amino acidsequences may be identical except for an amino acid substitution in nomore than 1, 2, 3, 4 or 5 amino acid positions. An antibody having VHand VL regions having high (i.e., 80% or greater) identity to the VH andVL regions of those described in Table 1 can be obtained by mutagenesis(e.g., site-directed or PCR-mediated mutagenesis) of nucleic acidmolecules encoding SEQ ID NOs: 13, 38, 58, 78, 98, 118, 118, or 138 andSEQ ID NOs: 23, 48, 68, 88, 108, 128, or 148, respectively, followed bytesting of the encoded altered antibody for retained function using thefunctional assays described herein.

In other embodiments, the full-length heavy chain and/or full-lengthlight chain amino acid sequences may be 50% 60%, 70%, 80%, 90%, 95%,96%, 97%, 98% or 99% identical to the sequences set forth in Table 1. Anantibody having a full-length heavy chain and full-length light chainhaving high (i.e., 80% or greater) identity to the full-length heavychains of any of SEQ ID NOs: 15, 28, 40, 60, 80, 100, 120, or 140, andfull-length light chains of any of SEQ ID NOs: 25, 25, 50, 70, 90, 110,130, or 150, can be obtained by mutagenesis (e.g., site-directed orPCR-mediated mutagenesis) of nucleic acid molecules encoding suchpolypeptides, followed by testing of the encoded altered antibody forretained function using the functional assays described herein.

In other embodiments, the full-length heavy chain and/or full-lengthlight chain nucleotide sequences may be 60%, 70%, 80%, 90%, 95%, 96%,97%, 98% or 99% identical to the sequences set forth in Table 1.

In other embodiments, the variable regions of heavy chain and/or thevariable regions of light chain nucleotide sequences may be 60%, 70%,80%, 90%, 95%, 96%, 97%, 98% or 99% identical to the sequences set forthin Table 1.

As used herein, the percent identity between the two sequences is afunction of the number of identical positions shared by the sequences(i.e., % identity equals number of identical positions/total number ofpositions×100), taking into account the number of gaps, and the lengthof each gap, which need to be introduced for optimal alignment of thetwo sequences. The comparison of sequences and determination of percentidentity between two sequences can be accomplished using a mathematicalalgorithm, as described in the non-limiting examples below.

Additionally or alternatively, the protein sequences of the presentinvention can further be used as a “query sequence” to perform a searchagainst public databases to, for example, identify related sequences.For example, such searches can be performed using the BLAST program(version 2.0) of Altschul, et al., 1990 J. Mol. Biol. 215:403-10.

Antibodies with Conservative Modifications

In certain embodiments, an antibody of the invention has a heavy chainvariable region comprising CDR1, CDR2, and CDR3 sequences and a lightchain variable region comprising CDR1, CDR2, and CDR3 sequences, whereinone or more of these CDR sequences have specified amino acid sequencesbased on the antibodies described herein or conservative modificationsthereof, and wherein the antibodies retain the desired functionalproperties of the ANGPTL4-binding antibodies of the invention.

Accordingly, the invention provides an isolated antibody, or a antigenbinding fragment thereof, consisting of a heavy chain variable regioncomprising CDR1, CDR2, and CDR3 sequences and a light chain variableregion comprising CDR1, CDR2, and CDR3 sequences, wherein: the heavychain variable region CDR1 amino acid sequences are selected from thegroup consisting of SEQ ID NOs: 7, 32, 52, 72, 92, 112, and 132, andconservative modifications thereof; the heavy chain variable region CDR2amino acid sequences are selected from the group consisting of SEQ IDNOs: 8, 33, 53, 73, 93, 113, and 133, and conservative modificationsthereof; the heavy chain variable region CDR3 amino acid sequences areselected from the group consisting of SEQ ID NOs: 9, 34, 54, 74, 94,114, and 134, and conservative modifications thereof; the light chainvariable regions CDR1 amino acid sequences are selected from the groupconsisting of SEQ ID NOs: 17, 42, 62, 82, 102, 122, and 142, andconservative modifications thereof; the light chain variable regionsCDR2 amino acid sequences are selected from the group consisting of SEQID NOs: 18, 43, 63, 83, 103, 123, and 143, and conservativemodifications thereof; the light chain variable regions of CDR3 aminoacid sequences are selected from the group consisting of SEQ ID NOs: 19,44, 64, 84, 104, 124, and 144, and conservative modifications thereof;and the antibody or antigen binding fragments thereof specifically bindsto ANGPTL4.

In other embodiments, the antibody of the invention is optimized forexpression in a mammalian cell has a full length heavy chain sequenceand a full length light chain sequence, wherein one or more of thesesequences have specified amino acid sequences based on the antibodiesdescribed herein or conservative modifications thereof, and wherein theantibodies retain the desired functional properties of the ANGPTL4binding antibodies of the invention. Accordingly, the invention providesan isolated antibody optimized for expression in a mammalian cellconsisting of a full-length heavy chain and a full-length light chainwherein the full length heavy chain has amino acid sequences selectedfrom the group of SEQ ID NOs: 15, 28, 40, 60, 80, 100, 120, and 140, andconservative modifications thereof; and the full length light chain hasamino acid sequences selected from the group of SEQ ID NOs: 25, 50, 70,90, 110, 130, and 150, and conservative modifications thereof; and theantibody specifically binds to ANGPTL4 (e.g., human and cynomolgusmonkey ANGPTL4).

Antibodies that Bind to the Same Epitope

The present invention provides antibodies that bind to the same epitopeas the ANGPTL4 binding antibodies described in Table 1. Additionalantibodies can therefore be identified based on their ability to compete(e.g., to competitively inhibit the binding of, in a statisticallysignificant manner) with other antibodies of the invention in ANGPTL4binding assays (such as those described in the Examples). The ability ofa test antibody to inhibit the binding of antibodies of the presentinvention to a ANGPTL4 protein demonstrates that the test antibody cancompete with that antibody for binding to ANGPTL4; such an antibody may,according to non-limiting theory, bind to the same or a related (e.g., astructurally similar or spatially proximal) epitope on the ANGPTL4protein as the antibody with which it competes. In a certain embodiment,the antibody that binds to the same epitope on ANGPTL4 as the antibodiesof the present invention is a humanized antibody. Such humanizedantibodies can be prepared and isolated as described herein. As usedherein, an antibody “competes” for binding when the competing antibodyinhibits ANGPTL4 binding of an antibody or antigen binding fragment ofthe invention by more than 50% (for example, 80%, 85%, 90%, 95%, 98% or99%) in the presence of an equimolar concentration of competingantibody.

In other embodiments the antibodies or antigen binding fragments of theinvention bind to one or more epitopes of ANGPTL4. In some embodiments,the epitopes to which the present antibodies or antigen bindingfragments bind are linear eptiopes. In other embodiments, the epitopesto which the present antibodies or antigen binding fragments bind arenon-linear, conformational eptiopes.

Engineered and Modified Antibodies

An antibody of the invention further can be prepared using an antibodyhaving one or more of the VH and/or VL sequences shown herein asstarting material to engineer a modified antibody, which modifiedantibody may have altered properties from the starting antibody. Anantibody can be engineered by modifying one or more residues within oneor both variable regions (i.e., VH and/or VL), for example within one ormore CDR regions and/or within one or more framework regions.Additionally or alternatively, an antibody can be engineered bymodifying residues within the constant region(s), for example to alterthe effector function(s) of the antibody.

One type of variable region engineering that can be performed is CDRgrafting. Antibodies interact with target antigens predominantly throughamino acid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al., 1998 Nature332:323-327; Jones, P. et al., 1986 Nature 321:522-525; Queen, C. etal., 1989 Proc. Natl. Acad., U.S.A. 86:10029-10033; U.S. Pat. No.5,225,539 to Winter, and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762and 6,180,370 to Queen et al.).

Accordingly, another embodiment of the invention pertains to an isolatedantibody, or an antigen binding fragment thereof, comprising a heavychain variable region comprising CDR1 sequences having an amino acidsequence selected from the group consisting of SEQ ID NOs: 7, 32, 52,72, 92, 112, and 132; CDR2 sequences having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 8, 33, 53, 73, 93,113, and 133; CDR3 sequences having an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 9, 34, 54, 74, 94, 114, and 134,respectively; and a light chain variable region having CDR1 sequenceshaving an amino acid sequence selected from the group consisting of SEQID NOs: 17, 42, 62, 82, 102, 122, and 142; CDR2 sequences having anamino acid sequence selected from the group consisting of SEQ ID NOs:18, 43, 63, 83, 103, 123, and 143; and CDR3 sequences consisting of anamino acid sequence selected from the group consisting of SEQ ID NOs:19, 44, 64, 84, 104, 124, and 144, respectively. Thus, such antibodiescontain the VH and VL CDR sequences of monoclonal antibodies, yet maycontain different framework sequences from these antibodies.

Such framework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chain variableregion genes can be found in the “VBase” human germline sequencedatabase (available on the world wide web at mrc-cpe.cam.ac.uk/vbase),as well as in Kabat, E. A., et al., 1991 Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242; Tomlinson, I. M., et al.,1992 J. Mol. Biol. 227:776-798; and Cox, J. P. L. et al., 1994 Eur. JImmunol. 24:827-836; the contents of each of which are expresslyincorporated herein by reference.

An example of framework sequences for use in the antibodies of theinvention are those that are structurally similar to the frameworksequences used by selected antibodies of the invention, e.g., consensussequences and/or framework sequences used by monoclonal antibodies ofthe invention. The VH CDR1, 2 and 3 sequences, and the VL CDR1, 2 and 3sequences, can be grafted onto framework regions that have the identicalsequence as that found in the germline immunoglobulin gene from whichthe framework sequence derive, or the CDR sequences can be grafted ontoframework regions that contain one or more mutations as compared to thegermline sequences. For example, it has been found that in certaininstances it is beneficial to mutate residues within the frameworkregions to maintain or enhance the antigen binding ability of theantibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and6,180,370 to Queen et al). Frameworks that can be utilized as scaffoldson which to build the antibodies and antigen binding fragments describedherein include, but are not limited to VH1A, VH1B, VH3, Vk1, VI2, andVk2. Additional frameworks are known in the art and may be found, forexample, in the vBase data base on the world wide web atvbase.mrc-cpe.cam.ac.uk/index.php?&MMN_position=1:1.

Accordingly, an embodiment of the invention relates to isolated ANGPTL4binding antibodies, or antigen binding fragments thereof, comprising aheavy chain variable region comprising an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 13, 38, 58, 78, 98, 118, and138, or an amino acid sequence having one, two, three, four or fiveamino acid substitutions, deletions or additions in the framework regionof such sequences, and further comprising a light chain variable regionhaving an amino acid sequence selected from the group consisting of SEQID NOs: 23, 48, 68, 88, 108, 128, and 148, or an amino acid sequencehaving one, two, three, four or five amino acid substitutions, deletionsor additions in the framework region of such sequences.

Another type of variable region modification is to mutate amino acidresidues within the VH and/or VL CDR1, CDR2 and/or CDR3 regions tothereby improve one or more binding properties (e.g., affinity) of theantibody of interest, known as “affinity maturation.” Site-directedmutagenesis or PCR-mediated mutagenesis can be performed to introducethe mutation(s) and the effect on antibody binding, or other functionalproperty of interest, can be evaluated in in vitro or in vivo assays asdescribed herein and provided in the Examples. Conservativemodifications (as discussed above) can be introduced. The mutations maybe amino acid substitutions, additions or deletions. Moreover, typicallyno more than one, two, three, four or five residues within a CDR regionare altered.

Accordingly, in another embodiment, the invention provides isolatedANGPTL4-binding antibodies, or antigen binding fragments thereof,consisting of a heavy chain variable region having a VH CDR1 regionconsisting of an amino acid sequence selected from the group having SEQID NOs: 7, 32, 52, 72, 92, 112, and 132, or an amino acid sequencehaving one, two, three, four or five amino acid substitutions, deletionsor additions as compared to SEQ ID NOs: 7, 32, 52, 72, 92, 112, 112, and132; a VH CDR2 region having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 8, 33, 53, 73, 93, 113, and 133, or anamino acid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to SEQ ID NOs: 8, 33,53, 73, 93, 113, and 133; a VH CDR3 region having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 9, 34, 54, 74, 94,114, 114, and 134, or an amino acid sequence having one, two, three,four or five amino acid substitutions, deletions or additions ascompared to SEQ ID NOs: 9, 34, 54, 74, 94, 114, and 134; a VL CDR1region having an amino acid sequence selected from the group consistingof SEQ ID NOs:17, 42, 62, 82, 102, 122, and 142, or an amino acidsequence having one, two, three, four or five amino acid substitutions,deletions or additions as compared to SEQ ID NOs: 17, 42, 62, 82, 102,122, and 142; a VL CDR2 region having an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 18, 43, 63, 83, 103, 123, and143, or an amino acid sequence having one, two, three, four or fiveamino acid substitutions, deletions or additions as compared to SEQ IDNOs: 18, 43, 63, 83, 103, 123, and 143; and a VL CDR3 region having anamino acid sequence selected from the group consisting of SEQ ID NOs:19, 44, 64, 84, 104, 124, and 144, or an amino acid sequence having one,two, three, four or five amino acid substitutions, deletions oradditions as compared to SEQ ID NOs: 19, 44, 64, 84, 104, 124, and 144.

Accordingly, in another embodiment, the invention provides isolatedANGPTL4-binding antibodies, or antigen binding fragments thereof,consisting of a heavy chain variable region having a VH CDR1 regionconsisting of an amino acid sequence selected from the group having SEQID NOs: 10, 35, 55, 75, 95, 115, and 135 or an amino acid sequencehaving one, two, three, four or five amino acid substitutions, deletionsor additions as compared to SEQ ID NOs: 10, 35, 55, 75, 95, 115, and135; a VH CDR2 region having an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 11, 36, 56, 76, 96, 116, and 136 or anamino acid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to SEQ ID NOs: 11, 36,56, 76, 96, 116, and 136; a VH CDR3 region having an amino acid sequenceselected from the group consisting of SEQ ID NOs: 12, 37, 57, 77, 97,117, and 137, or an amino acid sequence having one, two, three, four orfive amino acid substitutions, deletions or additions as compared to SEQID NOs: 12, 37, 57, 77, 97, 117, and 137; a VL CDR1 region having anamino acid sequence selected from the group consisting of SEQ ID NOs:20, 45, 65, 85, 105, 125, and 145, or an amino acid sequence having one,two, three, four or five amino acid substitutions, deletions oradditions as compared to SEQ ID NOs: 20, 45, 65, 85, 105, 125, and 145;a VL CDR2 region having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 21, 46, 66, 86, 106, 126, and 146, or an aminoacid sequence having one, two, three, four or five amino acidsubstitutions, deletions or additions as compared to SEQ ID NOs: 21, 46,66, 86, 106, 126, and 146; and a VL CDR3 region having an amino acidsequence selected from the group consisting of SEQ ID NOs: 22, 47, 67,87, 107, 127, and 147, or an amino acid sequence having one, two, three,four or five amino acid substitutions, deletions or additions ascompared to SEQ ID NOs: 22, 47, 67, 87, 107, 127, and 147.

Grafting Antigen-Binding Domains into Alternative Frameworks orScaffolds

A wide variety of antibody/immunoglobulin frameworks or scaffolds can beemployed so long as the resulting polypeptide includes at least onebinding region which specifically binds to ANGPTL4. Such frameworks orscaffolds include the 5 main idiotypes of human immunoglobulins, orfragments thereof, and include immunoglobulins of other animal species,preferably having humanized aspects. Single heavy-chain antibodies suchas those identified in camelids are of particular interest in thisregard. Novel frameworks, scaffolds and fragments continue to bediscovered and developed by those skilled in the art.

In one aspect, the invention pertains to generating non-immunoglobulinbased antibodies using non-immunoglobulin scaffolds onto which CDRs ofthe invention can be grafted. Known or future non-immunoglobulinframeworks and scaffolds may be employed, as long as they comprise abinding region specific for the target ANGPTL4 protein. Knownnon-immunoglobulin frameworks or scaffolds include, but are not limitedto, fibronectin (Compound Therapeutics, Inc., Waltham, Mass.), ankyrin(Molecular Partners AG, Zurich, Switzerland), domain antibodies(Domantis, Ltd., Cambridge, Mass., and Ablynx nv, Zwijnaarde, Belgium),lipocalin (Pieris Proteolab AG, Freising, Germany), small modularimmuno-pharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, Wash.),maxybodies (Avidia, Inc., Mountain View, Calif.), Protein A (AffibodyAG, Sweden), and affilin (gamma-crystallin or ubiquitin) (Scil ProteinsGmbH, Halle, Germany).

The fibronectin scaffolds are based on fibronectin type III domain(e.g., the tenth module of the fibronectin type III (10 Fn3 domain)).The fibronectin type III domain has 7 or 8 beta strands which aredistributed between two beta sheets, which themselves pack against eachother to form the core of the protein, and further containing loops(analogous to CDRs) which connect the beta strands to each other and aresolvent exposed. There are at least three such loops at each edge of thebeta sheet sandwich, where the edge is the boundary of the proteinperpendicular to the direction of the beta strands (see U.S. Pat. No.6,818,418). These fibronectin-based scaffolds are not an immunoglobulin,although the overall fold is closely related to that of the smallestfunctional antibody fragment, the variable region of the heavy chain,which comprises the entire antigen recognition unit in camel and llamaIgG. Because of this structure, the non-immunoglobulin antibody mimicsantigen binding properties that are similar in nature and affinity tothose of antibodies. These scaffolds can be used in a loop randomizationand shuffling strategy in vitro that is similar to the process ofaffinity maturation of antibodies in vivo. These fibronectin-basedmolecules can be used as scaffolds where the loop regions of themolecule can be replaced with CDRs of the invention using standardcloning techniques.

The ankyrin technology is based on using proteins with ankyrin derivedrepeat modules as scaffolds for bearing variable regions which can beused for binding to different targets. The ankyrin repeat module is a 33amino acid polypeptide consisting of two anti-parallel α-helices and aβ-turn. Binding of the variable regions is mostly optimized by usingribosome display.

Avimers are derived from natural A-domain containing protein such asLRP-1. These domains are used by nature for protein-protein interactionsand in human over 250 proteins are structurally based on A-domains.Avimers consist of a number of different “A-domain” monomers (2-10)linked via amino acid linkers. Avimers can be created that can bind tothe target antigen using the methodology described in, for example, U.S.Patent Application Publication Nos. 20040175756; 20050053973;20050048512; and 20060008844.

Affibody affinity ligands are small, simple proteins composed of athree-helix bundle based on the scaffold of one of the IgG-bindingdomains of Protein A. Protein A is a surface protein from the bacteriumStaphylococcus aureus. This scaffold domain consists of 58 amino acids,13 of which are randomized to generate affibody libraries with a largenumber of ligand variants (See e.g., U.S. Pat. No. 5,831,012). Affibodymolecules mimic antibodies, they have a molecular weight of 6 kDa,compared to the molecular weight of antibodies, which is 150 kDa. Inspite of its small size, the binding site of affibody molecules issimilar to that of an antibody.

Anticalins are products developed by the company Pieris ProteoLab AG.They are derived from lipocalins, a widespread group of small and robustproteins that are usually involved in the physiological transport orstorage of chemically sensitive or insoluble compounds. Several naturallipocalins occur in human tissues or body liquids. The proteinarchitecture is reminiscent of immunoglobulins, with hypervariable loopson top of a rigid framework. However, in contrast with antibodies ortheir recombinant fragments, lipocalins are composed of a singlepolypeptide chain with 160 to 180 amino acid residues, being justmarginally bigger than a single immunoglobulin domain. The set of fourloops, which makes up the binding pocket, shows pronounced structuralplasticity and tolerates a variety of side chains. The binding site canthus be reshaped in a proprietary process in order to recognizeprescribed target molecules of different shape with high affinity andspecificity. One protein of lipocalin family, the bilin-binding protein(BBP) of Pieris Brassicae has been used to develop anticalins bymutagenizing the set of four loops. One example of a patent applicationdescribing anticalins is in PCT Publication No. WO 199916873.

Affilin molecules are small non-immunoglobulin proteins which aredesigned for specific affinities towards proteins and small molecules.New affilin molecules can be very quickly selected from two libraries,each of which is based on a different human derived scaffold protein.Affilin molecules do not show any structural homology to immunoglobulinproteins. Currently, two affilin scaffolds are employed, one of which isgamma crystalline, a human structural eye lens protein and the other is“ubiquitin” superfamily proteins. Both human scaffolds are very small,show high temperature stability and are almost resistant to pH changesand denaturing agents. This high stability is mainly due to the expandedbeta sheet structure of the proteins. Examples of gamma crystallinederived proteins are described in WO200104144 and examples of“ubiquitin-like” proteins are described in WO2004106368.

Protein epitope mimetics (PEM) are medium-sized, cyclic, peptide-likemolecules (MW 1-2 kDa) mimicking beta-hairpin secondary structures ofproteins, the major secondary structure involved in protein-proteininteractions.

The present invention provides fully human antibodies that specificallybind to a ANGPTL4 protein. Compared to the chimeric or humanizedantibodies, the human ANGPTL4-binding antibodies of the invention havefurther reduced antigenicity when administered to human subjects.

Camelid Antibodies

Antibody proteins obtained from members of the camel and dromedary(Camelus bactrianus and Calelus dromaderius) family including new worldmembers such as llama species (Lama paccos, Lama glama and Lama vicugna)have been characterized with respect to size, structural complexity andantigenicity for human subjects. Certain IgG antibodies from this familyof mammals as found in nature lack light chains, and are thusstructurally distinct from the typical four chain quaternary structurehaving two heavy and two light chains, for antibodies from otheranimals. See PCT/EP93/02214 (WO 94/04678 published 3 Mar. 1994).

A region of the camelid antibody which is the small single variabledomain identified as VHH can be obtained by genetic engineering to yielda small protein having high affinity for a target, resulting in a lowmolecular weight antibody-derived protein known as a “camelid nanobody”.See U.S. Pat. No. 5,759,808 issued Jun. 2, 1998; see also Stijlemans, B.et al., 2004 J Biol Chem 279: 1256-1261; Dumoulin, M. et al., 2003Nature 424: 783-788; Pleschberger, M. et al. 2003 Bioconjugate Chem 14:440-448; Cortez-Retamozo, V. et al. 2002 Int J Cancer 89: 456-62; andLauwereys, M. et al. 1998 EMBO J 17: 3512-3520. Engineered libraries ofcamelid antibodies and antibody fragments are commercially available,for example, from Ablynx, Ghent, Belgium. As with other antibodies ofnon-human origin, an amino acid sequence of a camelid antibody can bealtered recombinantly to obtain a sequence that more closely resembles ahuman sequence, i.e., the nanobody can be “humanized”. Thus the naturallow antigenicity of camelid antibodies to humans can be further reduced.

The camelid nanobody has a molecular weight approximately one-tenth thatof a human IgG molecule, and the protein has a physical diameter of onlya few nanometers. One consequence of the small size is the ability ofcamelid nanobodies to bind to antigenic sites that are functionallyinvisible to larger antibody proteins, i.e., camelid nanobodies areuseful as reagents detect antigens that are otherwise cryptic usingclassical immunological techniques, and as possible therapeutic agents.Thus yet another consequence of small size is that a camelid nanobodycan inhibit as a result of binding to a specific site in a groove ornarrow cleft of a target protein, and hence can serve in a capacity thatmore closely resembles the function of a classical low molecular weightdrug than that of a classical antibody.

The low molecular weight and compact size further result in camelidnanobodies being extremely thermostable, stable to extreme pH and toproteolytic digestion, and poorly antigenic. Another consequence is thatcamelid nanobodies readily move from the circulatory system intotissues, and even cross the blood-brain barrier and can treat disordersthat affect nervous tissue. Nanobodies can further facilitated drugtransport across the blood brain barrier. See U.S. patent application20040161738 published Aug. 19, 2004. These features combined with thelow antigenicity to humans indicate great therapeutic potential.Further, these molecules can be fully expressed in prokaryotic cellssuch as E. coli and are expressed as fusion proteins with bacteriophageand are functional.

Accordingly, a feature of the present invention is a camelid antibody ornanobody having high affinity for ANGPTL4. In certain embodimentsherein, the camelid antibody or nanobody is naturally produced in thecamelid animal, i.e., is produced by the camelid following immunizationwith ANGPTL4 or a peptide fragment thereof, using techniques describedherein for other antibodies. Alternatively, the ANGPTL4-binding camelidnanobody is engineered, i.e., produced by selection for example from alibrary of phage displaying appropriately mutagenized camelid nanobodyproteins using panning procedures with ANGPTL4 as a target as describedin the examples herein. Engineered nanobodies can further be customizedby genetic engineering to have a half life in a recipient subject offrom 45 minutes to two weeks. In a specific embodiment, the camelidantibody or nanobody is obtained by grafting the CDRs sequences of theheavy or light chain of the human antibodies of the invention intonanobody or single domain antibody framework sequences, as described forexample in PCT/EP93/02214.

Bispecific Molecules and Multivalent Antibodies

In another aspect, the present invention features bispecific ormultispecific molecules comprising a ANGPTL4-binding antibody, or afragment thereof, of the invention. An antibody of the invention, orantigen-binding regions thereof, can be derivatized or linked to anotherfunctional molecule, e.g., another peptide or protein (e.g., anotherantibody or ligand for a receptor) to generate a bispecific moleculethat binds to at least two different binding sites or target molecules.The antibody of the invention may in fact be derivatized or linked tomore than one other functional molecule to generate multi-specificmolecules that bind to more than two different binding sites and/ortarget molecules; such multi-specific molecules are also intended to beencompassed by the term “bispecific molecule” as used herein. To createa bispecific molecule of the invention, an antibody of the invention canbe functionally linked (e.g., by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other bindingmolecules, such as another antibody, antibody fragment, peptide orbinding mimetic, such that a bispecific molecule results.

Accordingly, the present invention includes bispecific moleculescomprising at least one first binding specificity for ANGPTL4 and asecond binding specificity for a second target epitope. For example, thesecond target epitope is another epitope of ANGPTL4 different from thefirst target epitope.

Additionally, for the invention in which the bispecific molecule ismulti-specific, the molecule can further include a third bindingspecificity, in addition to the first and second target epitope.

In one embodiment, the bispecific molecules of the invention comprise asa binding specificity at least one antibody, or an antibody fragmentthereof, including, e.g., a Fab, Fab′, F(ab′)2, Fv, or a single chainFv. The antibody may also be a light chain or heavy chain dimer, or anyminimal fragment thereof such as a Fv or a single chain construct asdescribed in Ladner et al. U.S. Pat. No. 4,946,778.

Diabodies are bivalent, bispecific molecules in which VH and VL domainsare expressed on a single polypeptide chain, connected by a linker thatis too short to allow for pairing between the two domains on the samechain. The VH and VL domains pair with complementary domains of anotherchain, thereby creating two antigen binding sites (see e.g., Holliger etal., 1993 Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak et al., 1994Structure 2:1121-1123). Diabodies can be produced by expressing twopolypeptide chains with either the structure VHA-VLB and VHB-VLA (VH-VLconfiguration), or VLA-VHB and VLB-VHA (VL-VH configuration) within thesame cell. Most of them can be expressed in soluble form in bacteria.Single chain diabodies (scDb) are produced by connecting the twodiabody-forming polypeptide chains with linker of approximately 15 aminoacid residues (see Holliger and Winter, 1997 Cancer Immunol.Immunother., 45(3-4):128-30; Wu et al., 1996 Immunotechnology,2(1):21-36). scDb can be expressed in bacteria in soluble, activemonomeric form (see Holliger and Winter, 1997 Cancer Immunol.Immunother., 45(34): 128-30; Wu et al., 1996 Immunotechnology,2(1):21-36; Pluckthun and Pack, 1997 Immunotechnology, 3(2): 83-105;Ridgway et al., 1996 Protein Eng., 9(7):617-21). A diabody can be fusedto Fc to generate a “di-diabody” (see Lu et al., 2004 J. Biol. Chem.,279(4):2856-65).

Other antibodies which can be employed in the bispecific molecules ofthe invention are murine, chimeric and humanized monoclonal antibodies.

Bispecific molecules can be prepared by conjugating the constituentbinding specificities, using methods known in the art. For example, eachbinding specificity of the bispecific molecule can be generatedseparately and then conjugated to one another. When the bindingspecificities are proteins or peptides, a variety of coupling orcross-linking agents can be used for covalent conjugation. Examples ofcross-linking agents include protein A, carbodiimide,N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-I-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al., 1984 J. Exp. Med. 160:1686;Liu, M A et al., 1985 Proc. Natl. Acad. Sci. USA 82:8648). Other methodsinclude those described in Paulus, 1985 Behring Ins. Mitt. No. 78,118-132; Brennan et al., 1985 Science 229:81-83), and Glennie et al.,1987 J. Immunol. 139: 2367-2375). Conjugating agents are SATA andsulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill.).

When the binding specificities are antibodies, they can be conjugated bysulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In a particularly embodiment, the hinge region is modified tocontain an odd number of sulfhydryl residues, for example one, prior toconjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb×mAb, mAb×Fab,Fab×F(ab′)2 or ligand×Fab fusion protein. A bispecific molecule of theinvention can be a single chain molecule comprising one single chainantibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Bispecific molecules maycomprise at least two single chain molecules. Methods for preparingbispecific molecules are described for example in U.S. Pat. No.5,260,203; U.S. Pat. No. 5,455,030; U.S. Pat. No. 4,881,175; U.S. Pat.No. 5,132,405; U.S. Pat. No. 5,091,513; U.S. Pat. No. 5,476,786; U.S.Pat. No. 5,013,653; U.S. Pat. No. 5,258,498; and U.S. Pat. No.5,482,858.

Binding of the bispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (REA), FACS analysis, bioassay (e.g., growthinhibition), or Western Blot assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest.

In another aspect, the present invention provides multivalent compoundscomprising at least two identical or different antigen-binding portionsof the antibodies of the invention binding to ANGPTL4. Theantigen-binding portions can be linked together via protein fusion orcovalent or non covalent linkage. Alternatively, methods of linkage havebeen described for the bispecific molecules. Tetravalent compounds canbe obtained for example by cross-linking antibodies of the antibodies ofthe invention with an antibody that binds to the constant regions of theantibodies of the invention, for example the Fc or hinge region.

Trimerizing domain are described for example in Borean patent EP 1 01228061. Pentamerizing modules are described for example inPCT/EP97/05897.

Antibodies with Extended Half Life

The present invention provides for antibodies that specifically bind toANGPTL4 protein which have an extended half-life in vivo.

Many factors may affect a protein's half life in vivo. For examples,kidney filtration, metabolism in the liver, degradation by proteolyticenzymes (proteases), and immunogenic responses (e.g., proteinneutralization by antibodies and uptake by macrophages and dendriticcells). A variety of strategies can be used to extend the half life ofthe antibodies of the present invention. For example, by chemicallinkage to polyethyleneglycol (PEG), reCODE PEG, antibody scaffold,polysialic acid (PSA), hydroxyethyl starch (HES), albumin-bindingligands, and carbohydrate shields; by genetic fusion to proteins bindingto serum proteins, such as albumin, IgG, FcRn, and transferring; bycoupling (genetically or chemically) to other binding moieties that bindto serum proteins, such as nanobodies, Fabs, DARPins, avimers,affibodies, and anticalins; by genetic fusion to rPEG, albumin, domainof albumin, albumin-binding proteins, and Fc; or by incorporation intonanocarriers, slow release formulations, or medical devices.

To prolong the serum circulation of antibodies in vivo, inert polymermolecules such as high molecular weight PEG can be attached to theantibodies or a fragment thereof with or without a multifunctionallinker either through site-specific conjugation of the PEG to the N- orC-terminus of the antibodies or via epsilon-amino groups present onlysine residues. To pegylate an antibody, the antibody, or fragmentthereof, typically is reacted with polyethylene glycol (PEG), such as areactive ester or aldehyde derivative of PEG, under conditions in whichone or more PEG groups become attached to the antibody or antibodyfragment. The pegylation can be carried out by an acylation reaction oran alkylation reaction with a reactive PEG molecule (or an analogousreactive water-soluble polymer). As used herein, the term “polyethyleneglycol” is intended to encompass any of the forms of PEG that have beenused to derivatize other proteins, such as mono (C1-C10) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certainembodiments, the antibody to be pegylated is an aglycosylated antibody.Linear or branched polymer derivatization that results in minimal lossof biological activity will be used. The degree of conjugation can beclosely monitored by SDS-PAGE and mass spectrometry to ensure properconjugation of PEG molecules to the antibodies. Unreacted PEG can beseparated from antibody-PEG conjugates by size-exclusion or byion-exchange chromatography. PEG-derivatized antibodies can be testedfor binding activity as well as for in vivo efficacy using methodswell-known to those of skill in the art, for example, by immunoassaysdescribed herein. Methods for pegylating proteins are known in the artand can be applied to the antibodies of the invention. See for example,EP 0 154 316 by Nishimura et al. and EP 0 401 384 by Ishikawa et al.

Other modified pegylation technologies include reconstituting chemicallyorthogonal directed engineering technology (ReCODE PEG), whichincorporates chemically specified side chains into biosynthetic proteinsvia a reconstituted system that includes tRNA synthetase and tRNA. Thistechnology enables incorporation of more than 30 new amino acids intobiosynthetic proteins in E. coli, yeast, and mammalian cells. The tRNAincorporates a nonnative amino acid any place an amber codon ispositioned, converting the amber from a stop codon to one that signalsincorporation of the chemically specified amino acid.

Recombinant pegylation technology (rPEG) can also be used for serumhalflife extension. This technology involves genetically fusing a300-600 amino acid unstructured protein tail to an existingpharmaceutical protein. Because the apparent molecular weight of such anunstructured protein chain is about 15-fold larger than its actualmolecular weight, the serum halflife of the protein is greatlyincreased. In contrast to traditional PEGylation, which requireschemical conjugation and repurification, the manufacturing process isgreatly simplified and the product is homogeneous.

Polysialytion is another technology, which uses the natural polymerpolysialic acid (PSA) to prolong the active life and improve thestability of therapeutic peptides and proteins. PSA is a polymer ofsialic acid (a sugar). When used for protein and therapeutic peptidedrug delivery, polysialic acid provides a protective microenvironment onconjugation. This increases the active life of the therapeutic proteinin the circulation and prevents it from being recognized by the immunesystem. The PSA polymer is naturally found in the human body. It wasadopted by certain bacteria which evolved over millions of years to coattheir walls with it. These naturally polysialylated bacteria were thenable, by virtue of molecular mimicry, to foil the body's defense system.PSA, nature's ultimate stealth technology, can be easily produced fromsuch bacteria in large quantities and with predetermined physicalcharacteristics. Bacterial PSA is completely non-immunogenic, even whencoupled to proteins, as it is chemically identical to PSA in the humanbody.

Another technology includes the use of hydroxyethyl starch (“HES”)derivatives linked to antibodies. HES is a modified natural polymerderived from waxy maize starch and can be metabolized by the body'senzymes. HES solutions are usually administered to substitute deficientblood volume and to improve the rheological properties of the blood.Hesylation of an antibody enables the prolongation of the circulationhalf-life by increasing the stability of the molecule, as well as byreducing renal clearance, resulting in an increased biological activity.By varying different parameters, such as the molecular weight of HES, awide range of HES antibody conjugates can be customized.

Antibodies having an increased half-life in vivo can also be generatedintroducing one or more amino acid modifications (i.e., substitutions,insertions or deletions) into an IgG constant domain, or FcRn bindingfragment thereof (preferably a Fc or hinge Fc domain fragment). See,e.g., International Publication No. WO 98/23289; InternationalPublication No. WO 97/34631; and U.S. Pat. No. 6,277,375.

Further, antibodies can be conjugated to albumin (e.g., human serumalbumin; HSA) in order to make the antibody or antibody fragment morestable in vivo or have a longer half life in vivo. The techniques arewell-known in the art, see, e.g., International Publication Nos. WO93/15199, WO 93/15200, and WO 01/77137; and European Patent No. EP413,622. In addition, in the context of a bispecific antibody asdescribed above, the specificities of the antibody can be designed suchthat one binding domain of the antibody binds to ANGPTL4 while a secondbinding domain of the antibody binds to serum albumin, preferably HSA.

The strategies for increasing half life is especially useful innanobodies, fibronectin-based binders, and other antibodies or proteinsfor which increased in vivo half life is desired.

Antibody Conjugates

The present invention provides antibodies or fragments thereof thatspecifically bind to a ANGPTL4 protein recombinantly fused or chemicallyconjugated (including both covalent and non-covalent conjugations) to aheterologous protein or polypeptide (or fragment thereof, preferably toa polypeptide of at least 10, at least 20, at least 30, at least 40, atleast 50, at least 60, at least 70, at least 80, at least 90 or at least100 amino acids) to generate fusion proteins. In particular, theinvention provides fusion proteins comprising an antigen-bindingfragment of an antibody described herein (e.g., a Fab fragment, Fdfragment, Fv fragment, F(ab)2 fragment, a VH domain, a VH CDR, a VLdomain or a VL CDR) and a heterologous protein, polypeptide, or peptide.Methods for fusing or conjugating proteins, polypeptides, or peptides toan antibody or an antibody fragment are known in the art. See, e.g.,U.S. Pat. Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851,and 5,112,946; European Patent Nos. EP 307,434 and EP 367,166;International Publication Nos. WO 96/04388 and WO 91/06570; Ashkenazi etal., 1991, Proc. Natl. Acad. Sci. USA 88: 10535-10539; Zheng et al.,1995, J. Immunol. 154:5590-5600; and Vil et al., 1992, Proc. Natl. Acad.Sci. USA 89:11337-11341.

Additional fusion proteins may be generated through the techniques ofgene-shuffling, motif-shuffling, exon-shuffling, and/or codon-shuffling(collectively referred to as “DNA shuffling”). DNA shuffling may beemployed to alter the activities of antibodies of the invention orfragments thereof (e.g., antibodies or fragments thereof with higheraffinities and lower dissociation rates). See, generally, U.S. Pat. Nos.5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten etal., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, TrendsBiotechnol. 16(2):76-82; Hansson, et al., 1999, J. Mol. Biol.287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308-313(each of these patents and publications are hereby incorporated byreference in its entirety). Antibodies or fragments thereof, or theencoded antibodies or fragments thereof, may be altered by beingsubjected to random mutagenesis by error-prone PCR, random nucleotideinsertion or other methods prior to recombination. A polynucleotideencoding an antibody or fragment thereof that specifically binds to aANGPTL4 protein may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc. of one or more heterologousmolecules.

Moreover, the antibodies or fragments thereof can be fused to markersequences, such as a peptide to facilitate purification. In preferredembodiments, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 EtonAvenue, Chatsworth, Calif., 91311), among others, many of which arecommercially available. As described in Gentz et al., 1989, Proc. Natl.Acad. Sci. USA 86:821-824, for instance, hexa-histidine provides forconvenient purification of the fusion protein. Other peptide tags usefulfor purification include, but are not limited to, the hemagglutinin(“HA”) tag, which corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson et al., 1984, Cell 37:767), and the “flag”tag.

In other embodiments, antibodies of the present invention or fragmentsthereof conjugated to a diagnostic or detectable agent. Such antibodiescan be useful for monitoring or prognosing the onset, development,progression and/or severity of a disease or disorder as part of aclinical testing procedure, such as determining the efficacy of aparticular therapy. Such diagnosis and detection can accomplished bycoupling the antibody to detectable substances including, but notlimited to, various enzymes, such as, but not limited to, horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; prosthetic groups, such as, but not limited to,streptavidinlbiotin and avidin/biotin; fluorescent materials, such as,but not limited to, umbelliferone, fluorescein, fluoresceinisothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; luminescent materials, such as, but notlimited to, luminol; bioluminescent materials, such as but not limitedto, luciferase, luciferin, and aequorin; radioactive materials, such as,but not limited to, iodine (131I, 125I, 123I, and 121I), carbon (14C),sulfur (35S), tritium (3H), indium (115In, 113In, 112In, and 111In),technetium (99Tc), thallium (201Ti), gallium (68Ga, 67Ga), palladium(103Pd), molybdenum (99Mo), xenon (133Xe), fluorine (18F), 153Sm, 177Lu,159Gd, 149Pm, 140La, 175Yb, 166Ho, 90Y, 47Sc, 186Re, 188Re, 142 Pr,105Rh, 97Ru, 68Ge, 57Co, 65Zn, 85Sr, 32P, 153Gd, 169Yb, 51Cr, 54Mn,75Se, 113Sn, and 117Tin; and positron emitting metals using variouspositron emission tomographies, and noradioactive paramagnetic metalions.

The present invention further encompasses uses of antibodies orfragments thereof conjugated to a therapeutic moiety. An antibody orfragment thereof may be conjugated to a therapeutic moiety such as acytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent ora radioactive metal ion, e.g., alpha-emitters. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells.

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety or drug moiety that modifies a given biologicalresponse. Therapeutic moieties or drug moieties are not to be construedas limited to classical chemical therapeutic agents. For example, thedrug moiety may be a protein, peptide, or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, cholera toxin, ordiphtheria toxin; a protein such as tumor necrosis factor, α-interferon,β-interferon, nerve growth factor, platelet derived growth factor,tissue plasminogen activator, an apoptotic agent, an anti-angiogenicagent; or, a biological response modifier such as, for example, alymphokine.

Moreover, an antibody can be conjugated to therapeutic moieties such asa radioactive metal ion, such as alph-emiters such as 213Bi ormacrocyclic chelators useful for conjugating radiometal ions, includingbut not limited to, 131In, 131LU, 131Y, 131Ho, 131Sm, to polypeptides.In certain embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) whichcan be attached to the antibody via a linker molecule. Such linkermolecules are commonly known in the art and described in Denardo et al.,1998, Clin Cancer Res. 4(10):2483-90; Peterson et al., 1999, Bioconjug.Chem. 10(4):553-7; and Zimmerman et al., 1999, Nucl. Med. Biol.26(8):943-50, each incorporated by reference in their entireties.

Techniques for conjugating therapeutic moieties to antibodies are wellknown, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies 84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982,Immunol. Rev. 62:119-58.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Methods of Producing Antibodies of the Invention

Nucleic Acids Encoding the Antibodies

The invention provides substantially purified nucleic acid moleculeswhich encode polypeptides comprising segments or domains of theANGPTL4-binding antibody chains described above. Some of the nucleicacids of the invention comprise the nucleotide sequence encoding theheavy chain variable region shown in SEQ ID NO: 13, 38, 58, 78, 98, 118,or 138, and/or the nucleotide sequence encoding the light chain variableregion shown in SEQ ID NO: 23, 48, 68, 88, 108, 128, or 148. In aspecific embodiment, the nucleic acid molecules are those identified inTable 1. Some other nucleic acid molecules of the invention comprisenucleotide sequences that are substantially identical (e.g., at least65, 80%, 95%, or 99%) to the nucleotide sequences of those identified inTable 1. When expressed from appropriate expression vectors,polypeptides encoded by these polynucleotides are capable of exhibitingANGPTL4 antigen binding capacity.

Also provided in the invention are polynucleotides which encode at leastone CDR region and usually all three CDR regions from the heavy or lightchain of the ANGPTL4-binding antibody set forth above. Some otherpolynucleotides encode all or substantially all of the variable regionsequence of the heavy chain and/or the light chain of theANGPTL4-binding antibody set forth above. Because of the degeneracy ofthe code, a variety of nucleic acid sequences will encode each of theimmunoglobulin amino acid sequences.

The nucleic acid molecules of the invention can encode both a variableregion and a constant region of the antibody. Some of nucleic acidsequences of the invention comprise nucleotides encoding a heavy chainsequence that is substantially identical (e.g., at least 80%, 90%, or99%) to the heavy chain sequence set forth in SEQ ID NO: 15, 28, 40, 60,80, 100, 120, and 140. Some other nucleic acid sequences comprisingnucleotide encoding a light chain sequence that is substantiallyidentical (e.g., at least 80%, 90%, or 99%) to the light chain sequenceset forth in SEQ ID NO: 25, 50, 70, 90, 110, 130, and 150.

The polynucleotide sequences can be produced by de novo solid-phase DNAsynthesis or by PCR mutagenesis of an existing sequence (e.g., sequencesas described in the Examples below) encoding a ANGPTL4-binding antibodyor its binding fragment. Direct chemical synthesis of nucleic acids canbe accomplished by methods known in the art, such as the phosphotriestermethod of Narang et al., 1979, Meth. Enzymol. 68:90; the phosphodiestermethod of Brown et al., Meth. Enzymol. 68:109, 1979; thediethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:1859,1981; and the solid support method of U.S. Pat. No. 4,458,066.Introducing mutations to a polynucleotide sequence by PCR can beperformed as described in, e.g., PCR Technology: Principles andApplications for DNA Amplification, H. A. Erlich (Ed.), Freeman Press,NY, N.Y., 1992; PCR Protocols: A Guide to Methods and Applications,Innis et al. (Ed.), Academic Press, San Diego, Calif., 1990; Mattila etal., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods andApplications 1:17, 1991.

Also provided in the invention are expression vectors and host cells forproducing the ANGPTL4-binding antibodies described above. Variousexpression vectors can be employed to express the polynucleotidesencoding the ANGPTL4-binding antibody chains or binding fragments. Bothviral-based and nonviral expression vectors can be used to produce theantibodies in a mammalian host cell. Nonviral vectors and systemsinclude plasmids, episomal vectors, typically with an expressioncassette for expressing a protein or RNA, and human artificialchromosomes (see, e.g., Harrington et al., Nat Genet 15:345, 1997). Forexample, nonviral vectors useful for expression of the ANGPTL4-bindingpolynucleotides and polypeptides in mammalian (e.g., human) cellsinclude pThioHis A, B & C, pcDNA3.1/His, pEBVHis A, B & C, (Invitrogen,San Diego, Calif.), MPSV vectors, and numerous other vectors known inthe art for expressing other proteins. Useful viral vectors includevectors based on retroviruses, adenoviruses, adenoassociated viruses,herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barrvirus, vaccinia virus vectors and Semliki Forest virus (SFV). See, Brentet al., supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeldet al., Cell 68:143, 1992.

The choice of expression vector depends on the intended host cells inwhich the vector is to be expressed. Typically, the expression vectorscontain a promoter and other regulatory sequences (e.g., enhancers) thatare operably linked to the polynucleotides encoding a ANGPTL4-bindingantibody chain or fragment. In some embodiments, an inducible promoteris employed to prevent expression of inserted sequences except underinducing conditions. Inducible promoters include, e.g., arabinose, lacZ,metallothionein promoter or a heat shock promoter. Cultures oftransformed organisms can be expanded under noninducing conditionswithout biasing the population for coding sequences whose expressionproducts are better tolerated by the host cells. In addition topromoters, other regulatory elements may also be required or desired forefficient expression of a ANGPTL4-binding antibody chain or fragment.These elements typically include an ATG initiation codon and adjacentribosome binding site or other sequences. In addition, the efficiency ofexpression may be enhanced by the inclusion of enhancers appropriate tothe cell system in use (see, e.g., Scharf et al., Results Probl. CellDiffer. 20:125, 1994; and Bittner et al., Meth. Enzymol., 153:516,1987). For example, the SV40 enhancer or CMV enhancer may be used toincrease expression in mammalian host cells.

The expression vectors may also provide a secretion signal sequenceposition to form a fusion protein with polypeptides encoded by insertedANGPTL4-binding antibody sequences. More often, the insertedANGPTL4-binding antibody sequences are linked to a signal sequencesbefore inclusion in the vector. Vectors to be used to receive sequencesencoding ANGPTL4-binding antibody light and heavy chain variable domainssometimes also encode constant regions or parts thereof. Such vectorsallow expression of the variable regions as fusion proteins with theconstant regions thereby leading to production of intact antibodies orfragments thereof. Typically, such constant regions are human.

The host cells for harboring and expressing the ANGPTL4-binding antibodychains can be either prokaryotic or eukaryotic. E. coli is oneprokaryotic host useful for cloning and expressing the polynucleotidesof the present invention. Other microbial hosts suitable for use includebacilli, such as Bacillus subtilis, and other enterobacteriaceae, suchas Salmonella, Serratia, and various Pseudomonas species. In theseprokaryotic hosts, one can also make expression vectors, which typicallycontain expression control sequences compatible with the host cell(e.g., an origin of replication). In addition, any number of a varietyof well-known promoters will be present, such as the lactose promotersystem, a tryptophan (trp) promoter system, a beta-lactamase promotersystem, or a promoter system from phage lambda. The promoters typicallycontrol expression, optionally with an operator sequence, and haveribosome binding site sequences and the like, for initiating andcompleting transcription and translation. Other microbes, such as yeast,can also be employed to express ANGPTL4-binding polypeptides of theinvention. Insect cells in combination with baculovirus vectors can alsobe used.

In some preferred embodiments, mammalian host cells are used to expressand produce the ANGPTL4-binding polypeptides of the present invention.For example, they can be either a hybridoma cell line expressingendogenous immunoglobulin genes (e.g., the 1D6.C9 myeloma hybridomaclone as described in the Examples) or a mammalian cell line harboringan exogenous expression vector (e.g., the SP2/0 myeloma cellsexemplified below). These include any normal mortal or normal orabnormal immortal animal or human cell. For example, a number ofsuitable host cell lines capable of secreting intact immunoglobulinshave been developed including the CHO cell lines, various Cos celllines, HeLa cells, myeloma cell lines, transformed B-cells andhybridomas. The use of mammalian tissue cell culture to expresspolypeptides is discussed generally in, e.g., Winnacker, FROM GENES TOCLONES, VCH Publishers, N.Y., N.Y., 1987. Expression vectors formammalian host cells can include expression control sequences, such asan origin of replication, a promoter, and an enhancer (see, e.g., Queen,et al., Immunol. Rev. 89:49-68, 1986), and necessary processinginformation sites, such as ribosome binding sites, RNA splice sites,polyadenylation sites, and transcriptional terminator sequences. Theseexpression vectors usually contain promoters derived from mammaliangenes or from mammalian viruses. Suitable promoters may be constitutive,cell type-specific, stage-specific, and/or modulatable or regulatable.Useful promoters include, but are not limited to, the metallothioneinpromoter, the constitutive adenovirus major late promoter, thedexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP polIIIpromoter, the constitutive MPSV promoter, the tetracycline-inducible CMVpromoter (such as the human immediate-early CMV promoter), theconstitutive CMV promoter, and promoter-enhancer combinations known inthe art.

Methods for introducing expression vectors containing the polynucleotidesequences of interest vary depending on the type of cellular host. Forexample, calcium chloride transfection is commonly utilized forprokaryotic cells, whereas calcium phosphate treatment orelectroporation may be used for other cellular hosts. (See generallySambrook, et al., supra). Other methods include, e.g., electroporation,calcium phosphate treatment, liposome-mediated transformation, injectionand microinjection, ballistic methods, virosomes, immunoliposomes,polycation:nucleic acid conjugates, naked DNA, artificial virions,fusion to the herpes virus structural protein VP22 (Elliot and O'Hare,Cell 88:223, 1997), agent-enhanced uptake of DNA, and ex vivotransduction. For long-term, high-yield production of recombinantproteins, stable expression will often be desired. For example, celllines which stably express ANGPTL4-binding antibody chains or bindingfragments can be prepared using expression vectors of the inventionwhich contain viral origins of replication or endogenous expressionelements and a selectable marker gene. Following the introduction of thevector, cells may be allowed to grow for 1-2 days in an enriched mediabefore they are switched to selective media. The purpose of theselectable marker is to confer resistance to selection, and its presenceallows growth of cells which successfully express the introducedsequences in selective media. Resistant, stably transfected cells can beproliferated using tissue culture techniques appropriate to the celltype.

Generation of Monoclonal Antibodies of the Invention

Monoclonal antibodies (mAbs) can be produced by a variety of techniques,including conventional monoclonal antibody methodology e.g., thestandard somatic cell hybridization technique of Kohler and Milstein,1975 Nature 256: 495. Many techniques for producing monoclonal antibodycan be employed e.g., viral or oncogenic transformation of Blymphocytes.

Animal systems for preparing hybridomas include the murine, rat andrabbit systems. Hybridoma production in the mouse is a well establishedprocedure. Immunization protocols and techniques for isolation ofimmunized splenocytes for fusion are known in the art. Fusion partners(e.g., murine myeloma cells) and fusion procedures are also known.

Chimeric or humanized antibodies of the present invention can beprepared based on the sequence of a murine monoclonal antibody preparedas described above. DNA encoding the heavy and light chainimmunoglobulins can be obtained from the murine hybridoma of interestand engineered to contain non-murine (e.g., human) immunoglobulinsequences using standard molecular biology techniques. For example, tocreate a chimeric antibody, the murine variable regions can be linked tohuman constant regions using methods known in the art (see e.g., U.S.Pat. No. 4,816,567 to Cabilly et al.). To create a humanized antibody,the murine CDR regions can be inserted into a human framework usingmethods known in the art. See e.g., U.S. Pat. No. 5,225,539 to Winter,and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 toQueen et al.

In a certain embodiment, the antibodies of the invention are humanizedantibodies. Such humanized antibodies directed against ANGPTL4 can begenerated using transgenic or transchromosomic mice carrying parts ofthe human immune system rather than the mouse system. These transgenicand transchromosomic mice include mice referred to herein as HuMAb miceand KM mice, respectively, and are collectively referred to herein as“human Ig mice.”

The HuMAb Mouse® (Medarex, Inc.) contains human immunoglobulin geneminiloci that encode un-rearranged human heavy (μ and γ) and κ lightchain immunoglobulin sequences, together with targeted mutations thatinactivate the endogenous p and K chain loci (see e.g., Lonberg, et al.,1994 Nature 368(6474): 856-859). Accordingly, the mice exhibit reducedexpression of mouse IgM or κ, and in response to immunization, theintroduced human heavy and light chain transgenes undergo classswitching and somatic mutation to generate high affinity human IgGκmonoclonal (Lonberg, N. et al., 1994 supra; reviewed in Lonberg, N.,1994 Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. andHuszar, D., 1995 Intern. Rev. Immunol. 13: 65-93, and Harding, F. andLonberg, N., 1995 Ann. N. Y. Acad. Sci. 764:536-546). The preparationand use of HuMAb mice, and the genomic modifications carried by suchmice, is further described in Taylor, L. et al., 1992 Nucleic AcidsResearch 20:6287-6295; Chen, J. et at., 1993 International Immunology 5:647-656; Tuaillon et al., 1993 Proc. Natl. Acad. Sci. USA 94:3720-3724;Choi et al., 1993 Nature Genetics 4:117-123; Chen, J. et al., 1993 EMBOJ. 12: 821-830; Tuaillon et al., 1994 J. Immunol. 152:2912-2920; Taylor,L. et al., 1994 International Immunology 579-591; and Fishwild, D. etal., 1996 Nature Biotechnology 14: 845-851, the contents of all of whichare hereby specifically incorporated by reference in their entirety. Seefurther, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425;5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429;all to Lonberg and Kay; U.S. Pat. No. 5,545,807 to Surani et al.; PCTPublication Nos. WO 92103918, WO 93/12227, WO 94/25585, WO 97113852, WO98/24884 and WO 99/45962, all to Lonberg and Kay; and PCT PublicationNo. WO 01/14424 to Korman et al.

In another embodiment, human antibodies of the invention can be raisedusing a mouse that carries human immunoglobulin sequences on transgenesand transchomosomes such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. Such mice, referredto herein as “KM mice”, are described in detail in PCT Publication WO02/43478 to Ishida et al.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseANGPTL4-binding antibodies of the invention. For example, an alternativetransgenic system referred to as the Xenomouse (Abgenix, Inc.) can beused. Such mice are described in, e.g., U.S. Pat. Nos. 5,939,598;6,075,181; 6,114,598; 6, 150,584 and 6,162,963 to Kucherlapati et al.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseANGPTL4-binding antibodies of the invention. For example, mice carryingboth a human heavy chain transchromosome and a human light chaintranchromosome, referred to as “TC mice” can be used; such mice aredescribed in Tomizuka et al., 2000 Proc. Natl. Acad. Sci. USA97:722-727. Furthermore, cows carrying human heavy and light chaintranschromosomes have been described in the art (Kuroiwa et al., 2002Nature Biotechnology 20:889-894) and can be used to raiseANGPTL4-binding antibodies of the invention.

Humanized antibodies of the invention can also be prepared using phagedisplay methods for screening libraries of human immunoglobulin genes.Such phage display methods for isolating human antibodies areestablished in the art or described in the examples below. See forexample: U.S. Pat. Nos. 5,223,409; 5,403,484; and U.S. Pat. No.5,571,698 to Ladner et al.; U.S. Pat. Nos. 5,427,908 and 5,580,717 toDower et al.; U.S. Pat. Nos. 5,969,108 and 6,172,197 to McCafferty etal.; and U.S. Pat. Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313;6,582,915 and 6,593,081 to Griffiths et al.

Humanized antibodies of the invention can also be prepared using SCIDmice into which human immune cells have been reconstituted such that ahuman antibody response can be generated upon immunization. Such miceare described in, for example, U.S. Pat. Nos. 5,476,996 and 5,698,767 toWilson et al.

Framework or Fc Engineering

Engineered antibodies of the invention include those in whichmodifications have been made to framework residues within VH and/or VL,e.g., to improve the properties of the antibody. Typically suchframework modifications are made to decrease the immunogenicity of theantibody. For example, one approach is to “backmutate” one or moreframework residues to the corresponding germline sequence. Morespecifically, an antibody that has undergone somatic mutation maycontain framework residues that differ from the germline sequence fromwhich the antibody is derived. Such residues can be identified bycomparing the antibody framework sequences to the germline sequencesfrom which the antibody is derived. To return the framework regionsequences to their germline configuration, the somatic mutations can be“backmutated” to the germline sequence by, for example, site-directedmutagenesis. Such “backmutated” antibodies are also intended to beencompassed by the invention.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell-epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 20030153043 by Carr et al.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the invention may be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody of the invention may bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or be modified to alter its glycosylation, again toalter one or more functional properties of the antibody. Each of theseembodiments is described in further detail below. The numbering ofresidues in the Fc region is that of the EU index of Kabat.

In one embodiment, the hinge region of CH1 is modified such that thenumber of cysteine residues in the hinge region is altered, e.g.,increased or decreased. This approach is described further in U.S. Pat.No. 5,677,425 by Bodmer et al. The number of cysteine residues in thehinge region of CH1 is altered to, for example, facilitate assembly ofthe light and heavy chains or to increase or decrease the stability ofthe antibody.

In another embodiment, the Fc hinge region of an antibody is mutated todecrease the biological half-life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745 by Ward et al.

In another embodiment, the antibody is modified to increase itsbiological half-life. Various approaches are possible. For example, oneor more of the following mutations can be introduced: T252L, T254S,T256F, as described in U.S. Pat. No. 6,277,375 to Ward. Alternatively,to increase the biological half life, the antibody can be altered withinthe CH1 or CL region to contain a salvage receptor binding epitope takenfrom two loops of a CH2 domain of an Fc region of an IgG, as describedin U.S. Pat. Nos. 5,869,046 and 6,121,022 by Presta et al.

In yet other embodiments, the Fc region is altered by replacing at leastone amino acid residue with a different amino acid residue to alter theeffector functions of the antibody. For example, one or more amino acidscan be replaced with a different amino acid residue such that theantibody has an altered affinity for an effector ligand but retains theantigen-binding ability of the parent antibody. The effector ligand towhich affinity is altered can be, for example, an Fc receptor or the C1component of complement. This approach is described in further detail inU.S. Pat. Nos. 5,624,821 and 5,648,260, both by Winter et al.

In another embodiment, one or more amino acids selected from amino acidresidues can be replaced with a different amino acid residue such thatthe antibody has altered C1q binding and/or reduced or abolishedcomplement dependent cytotoxicity (CDC). This approach is described infurther detail in U.S. Pat. No. 6,194,551 by Idusogie et al.

In another embodiment, one or more amino acid residues are altered tothereby alter the ability of the antibody to fix complement. Thisapproach is described further in PCT Publication WO 94/29351 by Bodmeret al.

In yet another embodiment, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by modifying one or more amino acids. This approach isdescribed further in PCT Publication WO 00/42072 by Presta. Moreover,the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII and FcRn havebeen mapped and variants with improved binding have been described (seeShields, R. L. et al., 2001 J. Biol. Chem. 276:6591-6604).

In still another embodiment, the glycosylation of an antibody ismodified. For example, an aglycoslated antibody can be made (i.e., theantibody lacks glycosylation). Glycosylation can be altered to, forexample, increase the affinity of the antibody for “antigen’. Suchcarbohydrate modifications can be accomplished by, for example, alteringone or more sites of glycosylation within the antibody sequence. Forexample, one or more amino acid substitutions can be made that result inelimination of one or more variable region framework glycosylation sitesto thereby eliminate glycosylation at that site. Such aglycosylation mayincrease the affinity of the antibody for antigen. Such an approach isdescribed in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 byCo et al.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies of the invention to thereby produce an antibodywith altered glycosylation. For example, EP 1,176,195 by Hang et al.describes a cell line with a functionally disrupted FUT8 gene, whichencodes a fucosyl transferase, such that antibodies expressed in such acell line exhibit hypofucosylation. PCT Publication WO 03/035835 byPresta describes a variant CHO cell line, Lecl3 cells, with reducedability to attach fucose to Asn(297)-linked carbohydrates, alsoresulting in hypofucosylation of antibodies expressed in that host cell(see also Shields, R. L. et al., 2002 J. Biol. Chem. 277:26733-26740).PCT Publication WO 99/54342 by Umana et al. describes cell linesengineered to express glycoprotein-modifying glycosyl transferases(e.g., beta(1,4)-N acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al., 1999 Nat. Biotech. 17:176-180).

Methods of Engineering Altered Antibodies

As discussed above, the ANGPTL4-binding antibodies having VH and VLsequences or full length heavy and light chain sequences shown hereincan be used to create new ANGPTL4-binding antibodies by modifying fulllength heavy chain and/or light chain sequences, VH and/or VL sequences,or the constant region(s) attached thereto. Thus, in another aspect ofthe invention, the structural features of a ANGPTL4-binding antibody ofthe invention are used to create structurally related ANGPTL4-bindingantibodies that retain at least one functional property of theantibodies of the invention, such as binding to human ANGPTL4 and alsoinhibiting one or more functional properties of ANGPTL4 (e.g., inhibitANGPTL4 binding to the ANGPTL4 receptor, inhibit ANGPTL4-dependent cellproliferation).

For example, one or more CDR regions of the antibodies of the presentinvention, or mutations thereof, can be combined recombinantly withknown framework regions and/or other CDRs to create additional,recombinantly-engineered, ANGPTL4-binding antibodies of the invention,as discussed above. Other types of modifications include those describedin the previous section. The starting material for the engineeringmethod is one or more of the VH and/or VL sequences provided herein, orone or more CDR regions thereof. To create the engineered antibody, itis not necessary to actually prepare (i.e., express as a protein) anantibody having one or more of the VH and/or VL sequences providedherein, or one or more CDR regions thereof. Rather, the informationcontained in the sequence(s) is used as the starting material to createa “second generation” sequence(s) derived from the original sequence(s)and then the “second generation” sequence(s) is prepared and expressedas a protein.

Accordingly, in another embodiment, the invention provides a method forpreparing a ANGPTL4-binding antibody consisting of a heavy chainvariable region antibody sequence having a CDR1 sequence selected fromthe group consisting of SEQ ID NOs: 7, 32, 52, 72, 92, 112, and 132, aCDR2 sequence selected from the group consisting of SEQ ID NOs: 8, 33,53, 73, 93, 113, and 133, and/or a CDR3 sequence selected from the groupconsisting of SEQ ID NOs: 9, 34, 54, 74, 94, 114, and 134; and a lightchain variable region antibody sequence having a CDR1 sequence selectedfrom the group consisting of SEQ ID NOs: 17, 42, 62, 82, 102, 122, and142, a CDR2 sequence selected from the group consisting of SEQ ID NOs:18, 43, 63, 83, 103, 123, and 143, and/or a CDR3 sequence selected fromthe group consisting of SEQ ID NOs: 19, 44, 64, 84, 104, 124, and 144;altering at least one amino acid residue within the heavy chain variableregion antibody sequence and/or the light chain variable region antibodysequence to create at least one altered antibody sequence; andexpressing the altered antibody sequence as a protein.

Accordingly, in another embodiment, the invention provides a method forpreparing a ANGPTL4-binding antibody consisting of a heavy chainvariable region antibody sequence having a CDR1 sequence selected fromthe group consisting of SEQ ID NOs: 10, 35, 55, 75, 95, 115, and 135, aCDR2 sequence selected from the group consisting of SEQ ID NOs: 11, 36,56, 76, 96, 116, and 136, and/or a CDR3 sequence selected from the groupconsisting of SEQ ID NOs: 12, 37, 57, 77, 97, 117, and 137; and a lightchain variable region antibody sequence having a CDR1 sequence selectedfrom the group consisting of SEQ ID NOs: 20, 45, 65, 85, 105, 125, and145, a CDR2 sequence selected from the group consisting of SEQ ID NOs:21, 46, 66, 86, 106, 126, and 146, and/or a CDR3 sequence selected fromthe group consisting of SEQ ID NOs: 22, 47, 67, 87, 107, 127, and 147;altering at least one amino acid residue within the heavy chain variableregion antibody sequence and/or the light chain variable region antibodysequence to create at least one altered antibody sequence; andexpressing the altered antibody sequence as a protein.

Accordingly, in another embodiment, the invention provides a method forpreparing a ANGPTL4-binding antibody optimized for expression in amammalian cell consisting of: a full-length heavy chain antibodysequence having a sequence selected from the group of SEQ ID NOs: 15,28, 40, 60, 80, 100, 120, and 140; and a full length light chainantibody sequence having a sequence selected from the group of 25, 50,70, 90, 110, 130, and 150; altering at least one amino acid residuewithin the full length heavy chain antibody sequence and/or the fulllength light chain antibody sequence to create at least one alteredantibody sequence; and expressing the altered antibody sequence as aprotein. In one embodiment, the alteration of the heavy or light chainis in the framework region of the heavy or light chain.

The altered antibody sequence can also be prepared by screening antibodylibraries having fixed CDR3 sequences or minimal essential bindingdeterminants as described in US2005/0255552 and diversity on CDR1 andCDR2 sequences. The screening can be performed according to anyscreening technology appropriate for screening antibodies from antibodylibraries, such as phage display technology.

Standard molecular biology techniques can be used to prepare and expressthe altered antibody sequence. The antibody encoded by the alteredantibody sequence(s) is one that retains one, some or all of thefunctional properties of the ANGPTL4-binding antibodies describedherein, which functional properties include, but are not limited to,specifically binding to human, cynomolgus, rat, and/or mouse ANGPTL4;and the antibody inhibit ANGPTL4-dependent cell proliferation in a F36Eand/or Ba/F3-ANGPTL4R cell proliferation assay.

In certain embodiments of the methods of engineering antibodies of theinvention, mutations can be introduced randomly or selectively along allor part of an ANGPTL4-binding antibody coding sequence and the resultingmodified ANGPTL4-binding antibodies can be screened for binding activityand/or other functional properties as described herein. Mutationalmethods have been described in the art. For example, PCT Publication WO02/092780 by Short describes methods for creating and screening antibodymutations using saturation mutagenesis, synthetic ligation assembly, ora combination thereof. Alternatively, PCT Publication WO 03/074679 byLazar et al. describes methods of using computational screening methodsto optimize physiochemical properties of antibodies.

In certain embodiments of the invention antibodies have been engineeredto remove sites of deamidation. Deamidation is known to cause structuraland functional changes in a peptide or protein. Deamindation can resultin decreased bioactivity, as well as alterations in pharmacokinetics andantigenicity of the protein pharmaceutical. (Anal Chem. 2005 Mar. 1;77(5):1432-9).

In certain embodiments of the invention the antibodies have beenengineered to increase pl and inprove their drug-like properties. The plof a protein is a key determinant of the overall biophysical propertiesof a molecule. Antibodies that have low pls have been known to be lesssoluble, less stable, and prone to aggregation. Further, thepurification of antibodies with low pl is challenging and can beproblematic especially during scale-up for clinical use. Increasing thepl of the anti-ANGPTL4 antibodies, or Fabs, of the invention improvedtheir solubility, enabling the antibodies to be formulated at higherconcentrations (>100 mg/ml). Formulation of the antibodies at highconcentrations (e.g. >100 mg/ml) offers the advantage of being able toadminister higher doses of the antibodies into eyes of patients viaintravitreal injections, which in turn may enable reduced dosingfrequency, a significant advantage for treatment of chronic diseasesincluding cardiovascular disorders. Higher pls may also increase theFcRn-mediated recycling of the IgG version of the antibody thus enablingthe drug to persist in the body for a longer duration, requiring fewerinjections. Finally, the overall stability of the antibodies issignificantly improved due to the higher pl resulting in longershelf-life and bioactivity in vivo. Preferably, the pl is greater thanor equal to 8.2.

The functional properties of the altered antibodies can be assessedusing standard assays available in the art and/or described herein, suchas those set forth in the Examples (e.g., ELISAs).

Prophylactic and Therapeutic Uses

Antibodies that binds ANGPTL4 as described herein, can be used at atherapeutically useful concentration for the treatment of a disease ordisorder associated with increased ANGPTL4 levels and/or activity byadministering to a subject in need thereof an effective amount of theantibodies or antigen binding fragments of the invention. The presentinvention provides a method of treating ANGPTL4-associatedcardiovascular disorders by administering to a subject in need thereofan effective amount of the antibodies of the invention. The presentinvention provides a method of treating ANGPTL4-associatedcardiovascular disorders by administering to a subject in need thereofan effective amount of the antibodies of the invention.

The antibodies of the invention can be used, inter alia, to preventtreat, prevent, and improve ANGPTL4 associated conditions or disorders,including but not limited to any number of conditions or diseases inwhich the ANGPTL4 protein levels are aberrantly high and/or in which areduction of ANGPTL4 protein levels is sought. These conditions includebut are not limited to those involving lipid metabolism, such ashyperlipidemia, hyperlipoproteinemia and dyslipidemia, includingatherogenic dyslipidemia, diabetic dyslipidemia, hypertriglyceridemia(e.g., severe hypertriglyceridemia (e.g., with TG>1000 mg/dL),hypertriglyceridemia associated with obesity, and Type Vhypertriglyceridemia) hypercholesterolemia, chylomicronemia, mixeddyslipidemia (obesity, metabolic syndrome, diabetes, etc.),lipodystrophy, lipoatrophy, and other conditions caused by, e.g.,decreased LPL activity and/or LPL deficiency, decreased LDL receptoractivity and/or LDL receptor deficiency, altered ApoC2, ApoE deficiency,increased ApoB, increased production and/or decreased elimination ofvery low-density lipoprotein (VLDL), certain drug treatment (e.g.,glucocorticoid treatment-induced dyslipidemia), any geneticpredisposition, diet, life style, and the like.

Other ANGPTL4-associated diseases or disorders associated with orresulting from hyperlipidemia, hyperlipoproteinemia, and/ordyslipidemia, include, but are not limited to, cardiovascular diseasesor disorders, such as atherosclerosis, aneurysm, hypertension, angina,stroke, cerebrovascular diseases, congestive heart failure, coronaryartery diseases, myocardial infarction, peripheral vascular diseases,and the like; acute pancreatitis; nonalcoholic steatohepatitis (NASH);blood sugar disorders, such as diabetes; obesity, and the like.

The antibodies of the invention can also be used in combination withother agents for the prevention, treatment, or improvement of ANGPTL4associated disorders. For example, statin therapies may be used incombination with the ANGPTL4 antibodies and antigen binding fragments ofthe invention for the treatment of patients with triglyceride-relateddisorders.

Pharmaceutical Compositions

The invention provides pharmaceutical compositions comprising theANGPTL4-binding antibodies (intact or binding fragments) formulatedtogether with a pharmaceutically acceptable carrier. The compositionscan additionally contain one or more other therapeutic agents that aresuitable for treating or preventing, for example, cardiovasculardisorders. Pharmaceutically acceptable carriers enhance or stabilize thecomposition, or can be used to facilitate preparation of thecomposition. Pharmaceutically acceptable carriers include solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like that arephysiologically compatible.

A pharmaceutical composition of the present invention can beadministered by a variety of methods known in the art. The route and/ormode of administration vary depending upon the desired results. It ispreferred that administration be intravitreal, intravenous,intramuscular, intraperitoneal, or subcutaneous, or administeredproximal to the site of the target. The pharmaceutically acceptablecarrier should be suitable for intravitreal, intravenous, intramuscular,subcutaneous, parenteral, spinal or epidermal administration (e.g., byinjection or infusion). Depending on the route of administration, theactive compound, i.e., antibody, bispecific and multispecific molecule,may be coated in a material to protect the compound from the action ofacids and other natural conditions that may inactivate the compound.

The composition should be sterile and fluid. Proper fluidity can bemaintained, for example, by use of coating such as lecithin, bymaintenance of required particle size in the case of dispersion and byuse of surfactants. In many cases, it is preferable to include isotonicagents, for example, sugars, polyalcohols such as mannitol or sorbitol,and sodium chloride in the composition. Long-term absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate or gelatin.

Pharmaceutical compositions of the invention can be prepared inaccordance with methods well known and routinely practiced in the art.See, e.g., Remington: The Science and Practice of Pharmacy, MackPublishing Co., 20th ed., 2000; and Sustained and Controlled ReleaseDrug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., NewYork, 1978. Pharmaceutical compositions are preferably manufacturedunder GMP conditions. Typically, a therapeutically effective dose orefficacious dose of the ANGPTL4-binding antibody is employed in thepharmaceutical compositions of the invention. The ANGPTL4-bindingantibodies are formulated into pharmaceutically acceptable dosage formsby conventional methods known to those of skill in the art. Dosageregimens are adjusted to provide the optimum desired response (e.g., atherapeutic response). For example, a single bolus may be administered,several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. It is especially advantageous to formulateparenteral compositions in dosage unit form for ease of administrationand uniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subjects tobe treated; each unit contains a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention can be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level depends upon a variety of pharmacokinetic factors includingthe activity of the particular compositions of the present inventionemployed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors.

A physician or veterinarian can start doses of the antibodies of theinvention employed in the pharmaceutical composition at levels lowerthan that required to achieve the desired therapeutic effect andgradually increase the dosage until the desired effect is achieved. Ingeneral, effective doses of the compositions of the present invention,for the treatment of a cardiovascular disorders described herein varydepending upon many different factors, including means ofadministration, target site, physiological state of the patient, whetherthe patient is human or an animal, other medications administered, andwhether treatment is prophylactic or therapeutic. Treatment dosages needto be titrated to optimize safety and efficacy. For systemicadministration with an antibody, the dosage ranges from about 0.0001 to100 mg/kg, and more usually 0.01 to 15 mg/kg, of the host body weight.For intravitreal administration with an antibody, the dosage may rangefrom 0.1 mg/eye to 5 mg/eye. For example, 0.1 mg/ml, 0.2 mg/ml, 0.3mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.7 mg/ml, 0.8 mg/ml, 0.9 mg/ml,1.0 mg/ml, 1.1 mg/ml, 1.2 mg/ml, 1.3 mg/ml, 1.4 mg/ml, 1.5 mg/ml, 1.6mg/ml, 1.7 mg/ml, 1.8 mg/ml, 1.9 mg/ml, 2.0 mg/ml, 2.1 mg/ml, 2.2 mg/ml,2.3 mg/ml, 2.4 mg/ml, 2.5 mg/ml, 2.6 mg/ml, 2.7 mg/ml, 2.8 mg/ml, 2.9mg/ml, 3.0 mg/ml, 3.1 mg/ml, 3.2 mg/ml, 3.3 mg/ml, 3.4 mg/ml, 3.5 mg/ml,3.6 mg/ml, 3.7 mg/ml, 3.8 mg/ml, 3.9 mg/ml, 4.0 mg/ml, 4.1 mg/ml, 4.2mg/ml, 4.3 mg/ml, 4.4 mg/ml, 4.5 mg/ml, 4.6 mg/ml, 4.7 mg/ml, 4.8 mg/ml,4.9 mg/ml, or 5.0 mg/ml. An exemplary treatment regime entails systemicadministration once per every two weeks or once a month or once every 3to 6 months. An exemplary treatment regime entails systemicadministration once per every two weeks or once a month or once every 3to 6 months, or as needed (PRN).

Antibody is usually administered on multiple occasions. Intervalsbetween single dosages can be weekly, monthly or yearly. Intervals canalso be irregular as indicated by measuring blood levels ofANGPTL4-binding antibody in the patient. In addition alternative dosingintervals can be determined by a physician and administered monthly oras necessary to be efficacious. In some methods of systemicadministration, dosage is adjusted to achieve a plasma antibodyconcentration of 1-1000 μg/ml and in some methods 25-500 μg/ml.Alternatively, antibody can be administered as a sustained releaseformulation, in which case less frequent administration is required.Dosage and frequency vary depending on the half-life of the antibody inthe patient. In general, humanized antibodies show longer half life thanthat of chimeric antibodies and nonhuman antibodies. The dosage andfrequency of administration can vary depending on whether the treatmentis prophylactic or therapeutic. In prophylactic applications, arelatively low dosage is administered at relatively infrequent intervalsover a long period of time. Some patients continue to receive treatmentfor the rest of their lives. In therapeutic applications, a relativelyhigh dosage at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, and preferablyuntil the patient shows partial or complete amelioration of symptoms ofdisease. Thereafter, the patient can be administered a prophylacticregime.

EXAMPLES

The following examples are provided to further illustrate the inventionbut not to limit its scope. Other variants of the invention will bereadily apparent to one of ordinary skill in the art and are encompassedby the appended claims.

Example 1: Preparation of Purified Recombinant Human ANGPTL4 for Use asAntigen and in Antibody Characterization Experiments

A nucleic acid sequence encoding full-length human ANGPTL4 polypeptide(amino acids 26-406, matching NCBI sequence NM_139314.2) with N-terminalsignal peptide from human IgG-kappa (MKTFILLLWVLLLWVIFLLPGATA) (SEQ IDNO: 152), and C-terminal FLAG epitope (DYKDDDDKH) (SEQ ID NO: 153),hexahistidine purification tag (HHHHHH) (SEQ ID NO: 154), and Avi tag(i.e., BirA biotinylation sequence GGGLNDIFEAQKIEWHE) (SEQ ID NO: 155)was subcloned into the mammalian cell expression vector pRS5a togenerate the plasmid pRS-Ikk-hANGPTL4(26-406)-FLAG-6HIS-Avi containing a20 amino acid IKK signal sequence followed by amino acids 26-406 ofhuman ANGPTL4 with carboxyl-terminal Flag, 6HIS, and Avi tags (Table 2,SEQ ID NO: 156).

For some preparations, the following procedures were used to express,purify, and biotinylate human ANGPTL4 protein (Method 1):Suspension-adapted HEK293T cells were cultured in serum-free FreeStyle293 expression medium (Life Technologies, catalog number 12338-018) andtransfected with the plasmid pRS-Ikk-hANGPTL4(26-406)-FLAG-His6-Aviusing polyethyleneimine transfection reagent (Polysciences, catalognumber 23966). Five hours after transfection, heparin (Alfa Aesar,catalog number A16198) was added to the culture medium to a finalconcentration of 0.5 mg/ml. The cells were then cultured for 72-96 hoursand the cell culture supernatant was then harvested by centrifugation at4° C. and sterile-filtered using a 0.22 μm filter (Thermo, catalognumber 567-0010). The filtered cell culture supernatant was thenconcentrated to about 100 ml by tangential flow filtration. Theconcentrated supernatant was diluted to a volume of 1 Liter withTBS-glycerol buffer (50 mM Tris-HCl, 150 mM NaCl, and 15% glycerol, pH7.4), and the sample was concentrated to about 200 ml by tangential flowfiltration. Anti-Flag M2 agarose resin (Sigma, catalog number220102-177) pre-equilibrated with TBS-glycerol buffer was then added tothe sample, and the resulting solution was gently mixed for 1 hr at 4°C. The agarose resin was then washed 5 times with 25 ml TBS-glycerol,and the bound ANGPTL4 protein was eluted with 20 ml TBS-glycerolcontaining 0.2 mg/ml Flag peptide (Sigma, catalog number 220176-317).Peroxide-free Tween-20 (AppliChem, catalog number A1284,0025 was addedto the eluted protein solution to a final concentration of 0.1%, and theresulting solution was loaded onto a 5 ml HiTrap heparin column (GELifesciences, catalog number 17-0407-01) that was pre-equilibrated inTBS-glycerol containing 0.1% Tween-20 (Buffer A). The column was washedwith 50 ml Buffer A, followed by 50 ml Buffer A containing 300 mM NaCl.ANGPTL4 protein was then eluted with 20 ml Buffer A containing 600 mMNaCl. The eluted protein was concentrated using a centrifugalconcentrator with a 30 kDa molecular weight cutoff (Amicon Ultra,catalog number UFC903024). The purity of the purified ANGPTL4 protein asassessed by SDS-PAGE was >90%.

For some applications, ANGPTL4 proteins were site-specificallybiotinylated on the C-terminal Avi tag using 10 μg purifiedbiotin-protein ligase (BirA) (Avidity) per mg of ANGPTL4. The buffer wassupplemented with final concentrations of 10 mM ATP, 10 mM magnesiumacetate, and 0.5 M d-biotin. The reaction mixture was incubated for 2 hrat 30° C. and then overnight at 4° C., then loaded onto a HiLoadSuperdex 200 column (26 mm×600 mm) (GE Lifesciences, catalog number28-9893-36) that was equilibrated in Buffer A. Fractions from theSuperdex 200 column were analyzed using SDS-PAGE, and ANGPTL4 containingfractions were pooled and concentrated using a centrifugal concentrator.

For other preparations, the following procedures were used to express,purify, and biotinylate the human ANGPTL4 protein (Method 2). PlasmidpRS-Ikk-hANGPTL4(26-406)C-Flag6HisAvi was transiently transfected intoHEK293T cells using standard polyethylenimine (PEI) transfectionmethods. Cells were propagated in suspension culture in Freestyle 293expression media and transfection was carried out at 1×10⁶ cells/mlfinal cell concentration in 4 liters media using 1 liter flasks. Fivehours after transfection, heparin at a final concentration of 500 μg/mlwas added. Cells were grown at 37° C. and 5% CO₂ for 72 hr. Cells werethen pelleted by centrifugation, and the supernatant passed through a0.22 μm sterile filter. The clarified supernatant was concentrated andbuffer exchanged into Buffer B (50 mM Tris-HCl, 150 mM NaCl, 10%glycerol, 10 mM imidazole, pH 7.4) using tangential flow filtration(TFF). The concentrated sample was then passed over a 5 ml Ni-NTAaffinity column equilibrated with Buffer C (50 mM Tris.HCl, 150 mM NaCl,10% glycerol, 10 mM imidazole, 0.1% n-octyl-β-maltoside, pH 7.4). Afterloading the sample, the column was washed with the same buffer untilbaseline absorbance at 280 nm was reached. The bound ANGPTL4 protein wasthen eluted by using a gradient of imidazole (10 mM to 500 mM). Elutionfractions that contained human ANGPTL4 were pooled, concentrated usingan Amicon concentrator (molecular weight cut-off 10 kD),buffered-exchanged using PD-10 columns into storage buffer (50 mMTris-HCl, 150 mM NaCl, 15% Glycerol, pH 7.4), aliquoted, flash frozen inliquid nitrogen, and stored at −80° C. The purity of the purified humanANGPTL4 protein as assessed by SDS-PAGE was >90%.

For some applications, purified ANGPTL4 protein prepared as describedabove was site-specifically biotinylated as follows: purified protein in50 mM Bicine, pH 8.3 buffer at a final concentration of approximately 1mg/mL was incubated in the presence of 10 mM ATP, 10 mM magnesiumacetate, 0.1 mM biotin, and BirA biotin ligase (Avidity) at 30° C. for 1hr and then at 4° C. overnight. The protein was then concentrated usingan Amicon concentrator (molecular weight cut-off 10 kD),buffer-exchanged using PD-10 columns into storage buffer (50 mMTris-HCl, 150 mM NaCl, 15% glycerol, pH 7.4), aliquoted, flash frozen inliquid nitrogen, and stored at −80° C.

Example 2: Preparation of Purified Recombinant Human ANGPTL4 N-TerminalCoiled-Coil Domain Protein for Use in Antibody CharacterizationExperiments

Expression, purification, and biotinylation of the N-terminal coiledcoil domain of human ANGPTL4 (amino acids 26-161) was carried out usingessentially the same methods described for human full-length humanANGPTL4 in Example 1, Method 2. The sequence of the purified humanANGPTL4 N-terminal domain protein is shown in Table 2 (SEQ ID NO: 157).

TABLE 2 Amino acid sequences of human ANGPTL4(26-406)-FLAG-His6-Avi (the signal peptide is highlighted by underlining,and the N-terminal QP sequence after the signal peptideand the C-terminal FLAG-His6-Avi sequences are high-lighted with italics) SEQ ID Construct NO. Amino Acid Sequence Human 156MKTFILLLWVLLLWVIFLLPGATA QPGPVQSKSPRFASWDEMNVLAHGL ANGPTL4(26-LQLGQGLREHAERTRSQLSALERRLSACGSACQGTEGSTDLPLAPESRV 406)-FLAG-DPEVLHSLQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLRIQHLQSQFGL His6-AviLDHKHLDHEVAKPARRKRLPEMAQPVDPAHNVSRLHRLPRDCQELFQVGERQSGLFEIQPQGSPPFLVNCKMTSDGGWTVIQRRHDGSVDFNRPWEAYKAGFGDPHGEFWLGLEKVHSITGDRNSRLAVQLRDWDGNAELLQFSVHLGGEDTAYSLQLTAPVAGQLGATTVPPSGLSVPFSTWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNLNGQYFRSIPQQRQKLKKGIFWKTWRGRYYPLQATTMLIQPMAAEAASDYKDDDDKHHHHHHGGGLNDIFEAQKIEWHE Human 157MKTFILLLWVLLLWVIFLLPGATA QPGPVQSKSPRFASWDEMNVLAHGL ANGPTL4(26-LQLGQGLREHAERTRSQLSALERRLSACGSACQGTEGSTDLPLAPESRV 161)-FLAG-DPEVLHSLQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLRIQHLQSQFGL His6-AviLDHKHLDHEVAKPARRKRLPEMAQPVDPAHNVSRLHRLPRDYKDDDDKHHHHHHDYKDDDDKHHHHHHGGGLNDIFEAQKIEWHE

Example 3: Preparation and Screening of Monoclonal Antibodies

Recombinant human ANGPTL4 protein was prepared in-house as described inExample 1, and was used as immunogen for the generation of anti-ANGPTL4hybridoma clones. Bcl-2 transgenic mice were immunized with recombinanthuman ANGPTL4 according to a standard rapid immunization protocol.Hybridomas were generated by using a standard electrofusion-basedmethod.

CHO-K1PD cells stably expressing human ANGPTL4 fused to a transmembranedomain were generated using standard methods. Due to the presence of thetransmembrane domain, these cells display ANGPTL4 on the cell surface.Therefore, binding of antibodies to ANGPTL4 on the surface of thesecells can be detected using flow cytometry.

Hybridoma supernatants were screened by detecting binding of antibodiespresent in the supernatant to human ANGPTL4 expressed on the surface ofCHO-K1PD cells. Binding of antibodies to the cells was detected using afluorescently labeled anti-mouse secondary antibody and flow cytometry.Parental CHO-K1PD cells that do not express ANGPTL4 were used as anegative control. For hybridomas that bound to ANGPTL4, antibodies werepurified from cell supernatants using standard methods, and theresulting enriched supernatant was tested in the flow cytometry assaywith CHO-K1PD/ANGPTL4 and CHO-K1PD-Parental cells.

ANGPTL4 antibody titers in hybridoma supernatants were determined byusing a standard direct ELISA assay, in which recombinant human ANGPTL4protein was immobilized on the surface of the ELISA plate. Confirmedpositive hybridomas were subcloned, and the sequences of the monoclonalantibodies produced by these hybridomas was determined using standardmethods.

The monoclonal antibodies 14P18, 1761, 19C16 and 37P1 were subsequentlyshown to inhibit human ANGPTL4 mediated inhibition of human lipoproteinlipase using methods described in Example 7 below. The nucleotide andamino acid sequences of the heavy and light chain variable regions of14P18, 1761, 19C16 and 37P1 were determined using standard methods.

Example 4: Humanization of Monoclonal Antibodies

The process of humanization is well described in the art (Jones, et al1986, Queen, et al 1989, Riechmann, et al 1988, Verhoeyen, Milstein andWinter 1988). The term humanization is described as the transfer of theantigen-binding site of a non-human antibody, e.g., a murine derivedantibody, to a human acceptor framework, e.g., a human germline sequence(Retter, et al 2005). The main rationale for humanizing an antibody isto minimize the risk of developing an immunogenic response towards theantibody when the antibody is administered as a therapeutic in humans(Rebello, et al 1999).

The antigen-binding site comprises the complementary determining regions(CDRs) (Chothia and Lesk 1987, Kabat, et al 1991) and positions in theframework region of the variable domains (VL and VH) that directly orindirectly affect binding. Framework residues that may directly affectbinding can, for example, be found in the so called “outer” loop regionlocated between CDR2 and CDR3. Residues that indirectly affect bindingare for example found at so called Vernier Zones (Foote and Winter1992). They are thought to support CDR conformation. Those positionsoutside the CDRs are taken into account when choosing a suitableacceptor framework to minimize the number of deviations of the finalhumanized antibody to the human germline acceptor sequence in theframework regions.

Example 5: Antibody Sequence Optimization and Affinity Maturation

Certain amino acid sequence motifs are known to undergopost-translational modification (PTM) such as glycosylation (e.g.,NxS/T, where x is any amino acid except P), oxidation of free cysteines,deamidation (e.g., deamidation of N in NG sequences) or isomerization(e.g., at DG sequences). If present in the CDR regions, those motifs areideally removed by site-directed mutagenesis in order to increaseproduct homogeneity.

The process of affinity maturation is well described in the art. Amongmany display systems, phage display (Smith 1985) and display oneukaryotic cells such as yeast (Boder and Wittrup 1997) are the mostcommonly applied systems to select for antibody-antigen interaction.Advantages of these display systems are that they are suitable for awide range of antigens and that the selection stringency can be easilyadjusted. In phage display, scFv or Fab fragments can be displayed andin yeast display scFv, Fab or full-length IgG can be displayed. Thesecommonly applied methods allow selection of desired antibody variantsfrom larger libraries with diversities of more than 1×10⁷. Librarieswith smaller diversity (e.g. 1,000) may be screen by micro-expressionand ELISA.

Non-targeted or random antibody variant libraries can be generated forexample by error-prone PCR (Cadwell and Joyce 1994) and provide a verysimple, but sometimes limited approach. Another strategy is the CDRdirected diversification of an antibody candidate. One or more positionsin one or more CDRs can be targeted specifically using for exampledegenerate oligonucleotides (Thompson, et al 1996), trinucleotidemutagenesis (TRIM) (Kayushin, et al 1996), or any other approach knownto the art.

Example 6: Expression and Purification of Humanized Antibodies

DNA sequences coding for humanized VL and VH domains were ordered atGeneArt (Life Technologies, Inc., Regensburg, Germany) including codonoptimization for Homo Sapiens. Sequences coding for VL and VH domainswere subcloned by cutting and pasting from the GeneArt derived vectorsinto expression vectors suitable for expression and secretion bymammalian cells. The heavy and light chains were cloned into individualexpression vectors to allow co-transfection. Elements of the expressionvector include a promoter (Cytomegalovirus (CMV) enhancer-promoter), asignal sequence to facilitate secretion, a polyadenylation signal andtranscription terminator (from the Bovine Growth Hormone (BGH) gene), anelement allowing episomal replication and replication in prokaryotes(e.g., SV40 origin and ColE1 or others known in the art) and elements toallow selection (ampicillin resistance gene and zeocin marker).

Human Embryonic Kidney cells constitutively expressing the SV40 large Tantigen (HEK293-T ATCC11268) are one of the preferred host cell linesfor transient expression of humanized and/or optimized IgG proteins.Transfection is performed using PEI (Polyethylenimine, MW 25,000 linear,Polysciences, USA, catalog number 23966) as transfection reagent. ThePEI stock solution is prepared by carefully dissolving 1 g of PEI in 900ml cell culture grade water at room temperature (RT). To facilitatedissolution of PEI, the solution is acidified by addition of HCl to pH3-5, followed by neutralization with NaOH to a final pH of 7.05.Finally, the volume is adjusted to 1 L and the solution is filteredthrough a 0.22 μm filter, aliquotted and frozen at −80° C. until furtheruse. HEK 293T cells are cultivated using a Novartis proprietaryserum-free culture medium for transfection and propagation of the cells,and ExCell VPRO serum-free culture medium (SAFC Biosciences, USA, Cat.No. 24561C) as production/feed medium. Cells prepared for transienttransfections are cultivated in suspension culture. For small scale (<5L) transfections, cells are grown in Corning shake flasks (Corning,Tewksbury, Mass.) on an orbital shaker (100-120 rpm) in a humidifiedincubator at 5% CO₂ (seed flasks). Cells in the seed cultures should bemaintained in the exponential growth phase (cell densities between5×10⁵/ml and 3×10⁶/ml) and display a viability of >90% for transfection.For small scale (<5 L) transfection an aliquot of cells is taken out ofthe seed cultures and adjusted to 1.4×10⁶ cells/ml in 36% of the finalvolume with Novartis serum-free culture medium. The DNA solution(Solution 1:0.5 mg of heavy chain and 0.5 mg of light chain expressionplasmid for a 1 L transfection) is prepared by diluting the DNA to 1mg/I (final volume) in 7% of the final culture volume followed by gentlemixing. To prevent bacterial contamination, this solution is filteredusing a 0.22 μm filter (e.g., Millipore Stericup). Then 3 mg/L (finalvolume) of PEI solution is also diluted in 7% of final culture volumeand mixed gently (Solution 2). Both solutions are incubated for 5-10 minat room temperature (RT). Thereafter solution 2 is added to solution 1with gentle mixing and incubated for another 5-15 minutes at roomtemperature. The transfection mix is then added to the cells and thecultivation of cells is continued for 4 to 6 hours. Finally, theremaining 50% of total production volume is achieved by addition ofExCell® VPRO serum-free culture medium. The cell cultivation iscontinued for eleven days post-transfection.

The culture is harvested by centrifugation at 4500 rpm for 20 minutes at4° C. (Heraeus®, Multifuge 3 S-R, Thermo Scientific, Rockford, Ill.).The cell supernatant recovered is sterile-filtered through a stericupfilter (0.22 μm) and stored at 4° C. until further processing.Purification was performed on an “ÄKTA 100 Explorer Air” chromatographysystem at 4° C. in a cooling cabinet, using a freshly sanitized (0.25 MNaOH) HiTrap 5 ml Protein A MabSelect®SuRe column. The column wasequilibrated with 5 column volumes of phosphate buffered saline (PBS,Gibco, Life Technologies, Carlsbad, Calif.), and then thesterile-filtered supernatant was loaded at 4.0 ml/min. The column waswashed with 13 column volumes of PBS. Antibody was then eluted with 5column volumes of 50 mM citrate, 70 mM NaCl, pH 3.2. The eluate wascollected in 3 ml fractions and adjusted to pH 7 with 1 M Tris-HCl, pH10. The antibody containing fractions were pooled and sterile-filtered(Millipore Steriflip, 0.22 μm), the OD 280 nm was measured using aspectrophotometer (NanoDrop ND-1000), and the protein concentration wascalculated based on the OD 280 and the molar extinction coefficientwhich was calculated based on the protein sequence. The eluate wastested for aggregation by size exclusion chromatography with multi-anglelight scattering detector (SEC-MALS) and purity was assessed by gelelectrophoresis (SDS-PAGE), endotoxin assay (LAL) and mass spectrometry(MS). For the second purification step, if needed, antibody from thefirst purification was loaded onto a freshly sanitized (0.5 M NaOH) gelviltration column (Hi Load 16/60 Superdex 200, 120 mL, GE-Helthcare).The column was equilibrated with PBS and the run was done with PBSbuffer at a flow rate of 1 ml/min. The eluate was collected in 1.2 mlfractions. Antibody containing proteins were pooled, and the resultingpurified antibody analyzed as described for the first purification step.

Using the methods described above, the following humanized antibodieswere prepared, expressed and purified: NEG276, NEG276-LALA, NEG278,NEG310, NEG318, NEG318-LALA, NEG319, NEG313, and NEG315. The frameworkand parental antibodies for these humanized antibodies is shown in Table3, and the nucleotide and amino acid sequences are shown in Table 1. Allhumanized antibodies were prepared as human IgG1 antibodies, except forNEG276-LALA and NEG318-LALA, which were prepared using a modified Fcregion (human IgG1-LALA) in which the Leu234-Leu235 sequence in theheavy chain is replaced by Ala234-Ala235. Human IgG1-LALA antibodies areknown to have reduced antibody effector function compared to wild-typehuman IgG1 antibodies.

TABLE 3 Humanized ANGPTL4 antibodies of the invention. VH VL ParentalAntibody SEQ ID NO. SEQ ID NO. Framework Antibody NEG276 13 23hIgG1/kappa 19C16 NEG276- 13 23 hIgG1-LALA/ 19C16 LALA kappa NEG278 3848 hIgG1/kappa 19C16 NEG310 58 68 hIgG1/kappa 17B1 NEG313 78 88hIgG1/kappa 37P1 NEG315 98 108 hIgG1/kappa 37P1 NEG318 118 128hIgG1/kappa 14P18 NEG319 138 148 hIgG1/kappa 14P18

Example 7: Human Lipoprotein Lipase Assay

HEK 293T cells cultured in FreeStyle expression medium (Invitrogen) weretransfected with a mammalian expression plasmid encoding full-lengthhuman lipoprotein lipase (LPL) polypeptide (matching NCBI sequenceNM_000237.2) using a standard polyethyleneimine (PEI) transfectionmethod. At 24 hours after transfection, heparin was added to the culturemedium to a final concentration of 3 U/ml, to enhance release ofsecreted hLPL from the cell surface. At 60 hours post-transfection, theculture medium was collected, filtered using a 0.2 μm filter, andglycerol was added to a final concentration of 10% v/v. The resultingsolution was loaded onto a 5 ml Heparin Sepharose HiTrap column (GE)which had been pre-equilibrated with Buffer A (50 mM Tris-HCl, 200 mMNaCl, 10% v/v glycerol, pH7.2). The column was washed with Buffer A, andhuman LPL protein was then eluted with step gradients of 500 mM NaCl, 1MNaCl, and 2M NaCl in Buffer A. The purest and most catalytically activehuman LPL eluted at 2M NaCl. Aliquots of purified human LPL wereflash-frozen and stored at −80° C. until use.

The following protocol was used to assess the ability of antibodies ofthe invention to block ANGPTL4 inhibition of human lipoprotein lipase.The 384-well assay plate (Corning, catalog number 3573) and sample plate(Greiner Bio-one, catalog number 781201) were washed with 1% bovineserum albumin (BSA) (0.1 ml per well) for 30 min at room temperature.The plates were then washed twice with 0.05% Tween-20 solution.

ANGPTL4 antibody in 100 mM HEPES, pH 7.0 (20 μl per well, serialdilution with final assay concentrations ranging from 0.02 to 500 nM)was added to the sample plate, followed by 20 μl human ANGPTL4 protein(10 nM final assay concentration) in Assay Buffer (100 mM HEPES, 2 mMMgCl₂, pH 7.0), and the plate was incubated for 20 min at roomtemperature with gentle shaking. Lipoprotein lipase diluted in AssayBuffer (20 μl) was then added and the plate was incubated for 10 min atroom temperature with gentle shaking.

A Coupling Enzyme Mix containing acyl-coenzyme A oxidase (SekisuiDiagnostics, catalog number T-17), acyl-Coenzyme A synthetase (SekisuiDiagnostics, catalog number T-16), and horseradish peroxidase (SekisuiDiagnostics), ATP (Sigma, catalog number A7699) and coenzyme A (MPBiomedicals, catalog number 100493) in Assay Buffer was prepared.Catalase agarose beads (Sigma, catalog number C9284) were added to theCoupling Enzyme Mix, and the mixture was incubated at 4° C. for 30 minwith shaking, and the catalase agarose beads were then removed bycentrifugation.

Human VLDL (Millipore, catalog number LP1) was diluted in Assay Buffer,treated with catalase agarose beads for 30 min, and the beads wereremoved from the solution by centrifugation. Amplex Red (Invitrogen,catalog number A12222) in Assay Buffer was added to a concentration of33 μM.

To the solution in the sample plate containing LPL, ANGPTL4 and ANGPTL4antibody, Coupling Enzyme Mix (20 μl) was added, and 54 μl of theresulting solution was transferred to the assay plate. To initiate thelipoprotein lipase reaction, VLDL/Amplex Red solution (18 μl) was added,and resorufin fluorescence was monitored continuously for 30 minutesusing an EnVision multiwell plate reader (Perkin Elmer). Final assayconcentrations were: 9.4 nM ANGPTL4, ˜4 nM human lipoprotein lipase, 2.3μg/ml human VLDL, 0.75 mM ATP, 90 μM coenzyme A, 0.5 U/ml ACO, 1.25 U/mlACS, 1.2 U/ml HRP, and 10 μM Amplex Red.

The resulting resorufin fluorescence vs. time data was used to determinelipoprotein lipase enzyme activity (initial rate) for each sample.Control samples without LPL (background control) or without ANGPTL4 orANGPTL4 antibodies (LPL activity control) were used to normalize theenzyme activity, which was expressed as a percentage of the LPL controlactivity. Enzyme activity data for different ANGPTL4 antibodyconcentrations was plotted using GraphPad Prism software, and the datawas fitted to generate an EC₅₀ value for the ANGPTL4 antibody-mediatedincrease in lipoprotein lipase enzyme activity. In this assay, humanANGPTL4 at 10 nM concentration typically inhibited LPL activity by70-95%. ANGPTL4 antibodies of the invention dose-dependently reversedLPL inhibition by ANGPTL4. EC50 results from this assay are shown inTable 4. Representative data for selected antibodies of the invention isshown in FIG. 1.

TABLE 4 ANGPTL4 antibodies of the invention block human ANGPTL4inhibition of lipoprotein lipase (LPL). Human ANGPTL4 Antibody EC₅₀ (nM)NEG276 0.6 NEG276-LALA 0.7 NEG278 0.7 NEG310 1.6 NEG313 3 NEG315 3NEG318 1.4 NEG319 0.5

Example 8: Preparation of Cynomolgus Monkey, Mouse and Rat ANGPTL4 andHuman ANGPTL3 Proteins for Use in Antibody Characterization

The sequence of cynomolgus monkey ANGPTL4 was determined by amplifyingthe gene sequence from a cynomolgus monkey liver cDNA library (Biochain,catalog number C1534149-Cy, lot no B409051). The primers 5-UT-Cyno5′-ATCCCCGCTCCCAGGCTAC-3′ (SEQ ID NO: 158) and 3-UT-cyno5′-CAGCAAGGAGTGAAG-CTCCATGCC-3′ (SEQ ID NO: 159) were designed based onthe 5′ and 3′ untranslated regions of human ANGPTL4 cDNA (NCBI sequenceNM_139314.2). The gel purified PCR product was ligated intopCR4-Blunt-TOPO (Life Technologies, catalog number K2875-40) andsequenced. The cloned cynomolgus monkey ANGPTL4 cDNA encoded a 406 aminoacid protein with 95% homology to human ANGPTL4. The nucleic acidsequence encoding amino acids 26-406 of cynomolgus monkey ANGPTL4 wassubcloned into the mammalian expression vector pRS5, to produce theplasmid pRS-Ikk-cynoANGPTL4(26-406)-FLAG-6HIS-Avi, which has a 20 aminoacid Ikk signal sequence, amino acids 26-406 of cyno ANGPTL4, andcarboxyl-terminal FLAG, 6HIS, and Avi tags (SEQ ID NO: 160 in Table 5).

The cynomolgus monkey ANGPTL4(26-406)-FLAG-6HIS-Avi protein wasexpressed and purified using similar methods as described for humanANGPTL4(26-406)-FLAG-6HIS-Avi protein in Example 1. For someapplications, the purified cynomolgus monkey ANGPTL4 protein wassite-specifically biotinylated using a similar as described for humanANGPTL4 protein in Example 1. The purity of the purified cynomolgusmonkey ANGPTL4 protein as assessed by SDS-PAGE was >90%.

Cynomolgus monkey ANGPTL4(26-161)-FLAG-6HIS-Avi protein was preparedusing similar methods. The sequence of the cyno ANGPTL4(26-161) proteinencoded by its corresponding expression construct is shown in Table 5(SEQ ID NO: 161).

Expression, purification, and biotinylation of mouse ANGPTL4 (aminoacids 26-410) and rat ANGPTL4 (amino acids 24-405) was carried out usingessentially the same methods as described for human ANGPTL4 in Example1, Method 2. The sequences of the mouse and rat ANGPTL4 proteins encodedby the corresponding expression constructs is shown in Table 5 (SEQ IDNO: 162 and 163, respectively). The purity of the purified mouse ANGPTL4and rat ANGPTL4 proteins as assessed by SDS-PAGE was >90%.

ANGPTL3 (SEQ ID NO: 5, Table 1) is a human protein that is closelyrelated to ANGPTL4. To enable evaluation of possible binding ofantibodies of the invention to ANGPTL3, a humanANGPTL3(14-460)-FLAG-His-Avi protein (SEQ ID NO: 164, Table 5) wasexpressed, purified and biotinylated using similar methods as describedfor human ANGPTL4 in Example 1, Method 2.

TABLE 5 Amino acid sequences of cynomolgus monkey ANGPTL4(26-406)-FLAG-His6-Avi, mouse ANGPTL4(26-410)-FLAG-His6-Avi, rat ANGPTL4(24-405)-FLAG-His6-Avi, and human ANGPTL3(17-460)-FLAG-His-Avi(signal peptides are highlighted by underlining, and the N-terminal QP sequence after the signal peptide and the C-terminalFLAG-His6-Avi sequences are highlighted with italics) SEQ ID ConstructNO. Amino Acid Sequence Cynomolgus monkey 160 MKTFILLLWVLLLWVIFLLPGATAQPGPVQSKSPRFASWDEMNVLAH ANGPTL4(26-406)-GLLQLGQGLREHAERTRSQLNALERRLSACGSACQGTEGSTALPLAP FLAG-His6-AviESRVDPEVLHSLQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLRIQRLQSQVGLLDPKHLDHEVAKPARRKRRPEMAQPVDSAHNASRLHRLPRDCQELFEDGERQSGLFEIQPQGSPPFLVNCKMTSDGGWTVIQRRHDGSVDFNRPWEAYKAGFGDPQGEFWLGLEKVHSITGDRNSRLAVQLQDWDGNAESLQFSVHLGGEDTAYSLQLTEPVASQLGATTVPPSGLSVPFSTWDQDHDLRRDKNCAKSLSGGWWFGTCSHSNLNGQYFRSIPQQRQELKKGIFWKTWRGRYYPLQATTMLIQPTAAEAASDYKDDDDKHHHHHHGG GLNDIFEAQKIEWHECynomolgus monkey 161 MKTFILLLWVLLLWVIFLLPGATA QPGPVQSKSPRFASWDEMNVLAHANGPTL4(26-161)- GLLQLGQGLREHAERTRSQLNALERRLSACGSACQGTEGSTALPLAPFLAG-His6-Avi ESRVDPEVLHSLQTQLKAQNSRIQQLFHKVAQQQRHLEKQHLRIQRLQSQVGLLDPKHLDHEVAKPARRKRRPEMAQPVDSAHNASRLHRLPRDYKDDDDKHHHHHHGGGLNDIFEAQKIEWHE Mouse ANGPTL4(26- 162MKTFILLLWVLLLWVIFLLPGATA QPRPAQPEPPRFASWDEMNLLAH 410)-FLAG-His6-AviGLLQLGHGLREHVERTRGQLGALERRMAACGNACQGPKGKDAPFKDSEDRVPEGQTPETLQSLQTQLKAQNSKIQQLFQKVAQQQRYLSKQNLRIQNLQSQIDLLAPTHLDNGVDKTSRGKRLPKMTQLIGLTPNATHLHRPPRDCQELFQEGERHSGLFQIQPLGSPPFLVNCEMTSDGGWTVIQRRLNGSVDFNQSWEAYKDGFGDPQGEFWLGLEKMHSITGNRGSQLAVQLQDWDGNAKLLQFPIHLGGEDTAYSLQLTEPTANELGATNVSPNGLSLPFSTWDQDHDLRGDLNCAKSLSGGWWFGTCSHSNLNGQYFHSIPRQRQERKKGIFWKTWKGRYYPLQATTLLIQPMEATAASDYKDDDDKHHHH HHGGGLNDIFEAQKIEWHERat ANGPTL4(24- 163 MKTFILLLWVLLLWVIFLLPGATA QPQGRPAQPEPPRFASWDEMNLL405)-FLAG-His6-Avi AHGLLQLGHGLREHVERTRGQLGALERRMAACGNACQGPKGTDPKDRVPEGQAPETLQSLQTQLKAQNSKIQQLFQKVAQQQRYLSKQNLRIQNLQSQIDLLTPTHLDNGVDKTSRGKRLPKMAQLIGLTPNATRLHRPPRDCQELFQEGERHSGLFQIQPLGSPPFLVNCEMTSDGGWTVIQRRLNGSVDFNQSWEAYKDGFGDPQGEFWLGLEKMHSITGDRGSQLAVQLQDWDGNAKLLQFPIHLGGEDTAYSLQLTEPTANELGATNVSPNGLSLPFSTWDQDHDLRGDLNCAKSLSGGWWFGTCSHSNLNGQYFHSIPRQRQQRKKGIFWKTWKGRYYPLQATTLLIQPMEATAASDYKDDDDKHHHHHHG GGLNDIFEAQKIEWHETHuman ANGPTL3(17- 164 MKTFILLLWVLLLWVIFLLPGATA QPSRIDQDNSSFDSLSPEPKSRF460) AMLDDVKILANGLLQLGHGLKDFVHKTKGQINDIFQKLNIFDQSFYDLSLQTSEIKEEEKELRRTTYKLQVKNEEVKNMSLELNSKLESLLEEKILLQQKVKYLEEQLTNLIQNQPETPEHPEVTSLKTFVEKQDNSIKDLLQTVEDQYKQLNQQHSQIKEIENQLRRTSIQEPTEISLSSKPRAPRTTPFLQLNEIRNVKHDGIPAECTTIYNRGEHTSGMYAIRPSNSQVFHVYCDVISGSPWTLIQHRIDGSQNFNETWENYKYGFGRLDGEFWLGLEKIYSIVKQSNYVLRIELEDWKDNKHYIEYSFYLGNHETNYTLHLVAITGNVPNAIPENKDLVFSTWDHKAKGHFNCPEGYSGGWWWHDECGENNLNGKYNKPRAKSKPERRRGLSWKSQNGRLYSIKSTKMLIHPTDSESFEDYKDDDDKHHHHHHGGGLNDIFEAQKIEWHE

Example 9: Characterization of Antibody Binding Specificity by DirectELISA Assays

Direct ELISA assays were conducted to characterize antibody bindingspecificity of selected antibodies of the invention. The assay wasperformed as follows. A 384-well streptavidin-coated Meso ScaleDiscovery (MSD) plate was blocked by incubating the plate with 50 μLBlocking Buffer (PBS, pH 7.4, 5% w/v bovine serum albumin) per well at22° C. for 1 hour with constant shaking (600 rpm). The blocked MSD platewas then washed 3 times with Wash Buffer (PBS, pH 7.4 and 0.05% v/vTween-20) using a plate washer (BioTek). Following washing, biotinylatedhuman ANGPTL4 proteins diluted in Assay Buffer (PBS, pH 7.4 withoutCaCl₂ or MgCl₂, 0.5% w/v fatty acid-free bovine serum albumin and 0.02%v/v Tween-20) were immobilized on the surface by incubation at 1 nMconcentration (15 μL per well) at 22° C. for one hour: full length humanANGPTL4 (hANGPTL4), human ANGPTL4 coiled-coil domain (hANGPTL4-CCD) andfull length human ANGPTL3 (hANGPTL3). The plate was then washed 3 timesas described earlier. Antibody diluted to 1 nM concentration in AssayBuffer was then applied to the MSD plate (15 μL per well), and the platewas incubated for 1 hour at 22° C. with constant shaking (600 rpm).Bound antibodies were detected by adding 15 μL per well of a 1:500dilution of Sulfo-tagged goat anti-human IgG. The plate was thenincubated for one hour with constant shaking (600 rpm). The plate waswashed 3 times, and then 15 μL/well of 1×MSD read buffer T was added andthe plate was developed using a Sector Imager 6000 (Meso ScaleDiscovery). The data were transferred to Microsoft Excel for analysisand plotted using GraphPad Prism v6. These experiments showed that allof the antibodies of the invention tested in this assay bind tofull-length human ANGPTL4 and to the N-terminal domain of human ANGPTL4,and do not bind to full-length human ANGPTL3. An ANGPTL3-specificreference antibody was used as a positive control for the ANGPTL3binding assay (FIG. 2).

Example 10: Antibody Dissociation Constants Determination by SolutionEquilibrium Titration (SET) Assay

SET assays were performed as follows. In a 96-well polypropylene plate,a constant concentration of ANGPTL4 antibody (10 pM) was mixed withdifferent concentrations of non-biotinylated human, cyno, mouse, or ratfull-length ANPGLT4 protein, or human ANGPTL4 N-terminal domain protein(5-fold serial dilution ranging from 0.01 pM to 100 nM) in SET buffer(PBS, pH 7.4 without CaCl₂ or MgCl₂, 0.5% w/v bovine serum albumin(fatty acid free) and 0.02% v/v Tween-20). The final reaction volume was80 μL. The plate was sealed using an adhesive film and incubated at 22°C. for 14 hours with constant shaking (300 rpm). During the same periodof time, a 384-well streptavidin-coated Meso Scale Discovery (MSD) platewas blocked by incubating the plate with 50 μL blocking buffer (PBS, pH7.4, 5% w/v bovine serum albumin) per well at 4° C. The blocked MSDplate was washed 3 times with wash buffer (PBS, pH 7.4 and 0.05% v/vTween-20) using a plate washer (BioTek). Biotinylated ANGPTL4(full-length human, cyno, mouse, or rat ANGPTL4, or human ANGPTL4N-terminal domain) protein (1 nM, 15 μL per well) was immobilized on thesurface of the streptavidin-coated MSD plate by incubation at 22° C. forone hour with constant shaking (600 rpm). The plate was then washed 3times as described earlier.

The equilibrium binding reactions (15 μL per well) were applied to theMSD plate with immobilized ANGPTL4 and incubated for 20 min at 22° C.The unbound material was removed by washing the plate 3 times with washbuffer, and the captured antibody was detected by adding 15 μL per wellof a 1:500 dilution of Sulfo-tagged goat anti-human IgG (Meso ScaleDiscovery). The plate was then incubated for one hour with constantshaking (600 rpm). The plate was washed 3 times, and then 15 μL/well of1×MSD read buffer T was added and the plate was developed using a SectorImager 6000 (Meso Scale Discovery). The data were transferred toMicrosoft Excel for analysis and plotted using GraphPad Prism v6. TheK_(D) values were determined by fitting the data to the followingequation:

γ=(B _(max)/(C _(Ab)/2))*((C _(Ab)/2)−((((((C _(Ag) +C _(Ab))+K_(D))/2)-((((((C _(Ag) +C _(Ab))+K _(D))̂2)/4)−(C _(Ag) *C_(Ab)))̂0.5))̂2)/(2*C _(Ab)))),

where B_(max) is the signal when no ANGPTL4 protein is present insolution, C_(Ab) is the constant concentration of ANGPTL4 antibody insolution, C_(Ag) is the concentration of ANGPTL4 in solution, and K_(D)is the equilibrium dissociation constant. Equilibrium dissociationconstants determined using this method are shown in Table 6.

TABLE 6 Dissociation constants (K_(D)) for antibodies of the inventionbinding to ANGPTL4 proteins determined by Solution Equilibrium Titration(SET) assays Human Cyno Rat Mouse ANGPTL4 ANGPTL4 ANGPTL4 ANGPTL4Antibody (26-406) (26-406) (24-405) (26-410) (IgG) K_(D) (pM) K_(D) (pM)K_(D) (pM) K_(D) (pM) NEG276 24 14 >500* >500* NEG276- 8 7 >500* >500*LALA NEG278 15 20 >500* >500* NEG310 21 22 >500* >500* NEG313 915 >500* >500* NEG315 12 21 >500* >500* NEG318 9 17 >500* >500* NEG31916 8 >500* >500* Ref Ab* 17 4 517 194 *No binding signal was detectedwith the experimental conditions used, indicating a K_(D) value >500 pM.A K_(D) value of 517 pM was determined for binding of an ANGPTL4reference antibody to rat ANGPTL4.

Example 11: Antibody Binding Kinetics and Dissociation ConstantsDetermined by Octet Kinetic Binding Assay

Dissociation constants (K_(D)) were determined for selected antibodiesof the invention by using an Octet (ForteBio) kinetic binding assay.ForteBio 10× Kinetics Buffer (ForteBio, catalog number 18-5032) wasdiluted 10-fold with DPBS (Life Technologies, catalog number 14190-136),and the resulting solution was added (0.2 ml per well) to a 96-wellplate (Greiner, catalog number 65520). Streptavidin sensors (ForteBio,catalog number 18-5020) were immersed in the solution and equilibratedfor at least 10 min at room temperature. In a second 96-well plate(Greiner, catalog number 65520), sensors were washed in 1× Kineticsbuffer, and then immersed in 200 ul of 25 nM biotinylated human ANGPTL4or a biotinylated reference protein (for background substraction) for1000 sec at seconds at room temperature. The sensors were then washed in1× Kinetics buffer for 120 sec, and immersed in 200 μl of ANGPTL4antibody diluted in 1× Kinetics buffer at various concentrations (serial2-fold dilutions; the highest concentrations were 12.5 nM or 25 nM; thelowest concentrations ranged from 0.8 to 3.1 nM; 4-6 different antibodyconcentrations were used for each K_(D) determination), and antibodyassociation was monitored for 480 seconds. The sensors were thentransferred to a well containing 200 μl 1× Kinetics buffer, and antibodydissociation was monitored for 1200 seconds. Background-correctedassociation and dissociation curves were globally fitted by OctetSoftware (ForteBio) to generate association (k_(a)) and dissociation(k_(d)) rate constants, which in turn were used to calculate equilibriumdissociation constants (K_(D)). The resulting data for selectedantibodies of the invention is shown in Table 7 and Table 8.

TABLE 7 Human ANGPTL4(26-406) antibody dissociation constants (K_(D))determined by ForteBio kinetic binding assay Human ANGPTL4(26-406)Antibody k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (pM) NEG276 3.4 × 10⁵ 8.0 ×10⁻⁶ 23 NEG278 3.0 × 10⁵ 6.7 × 10⁻⁷ ≦17*  NEG310 2.2 × 10⁵ 1.1 × 10⁻⁵ 25NEG313 2.8 × 10⁵ 1.1 × 10⁻⁵ 40 NEG315 2.8 × 10⁵ 7.9 × 10⁻⁶ 29 NEG318 2.7× 10⁵ 1.2 × 10⁻⁵ 45 NEG319 2.8 × 10⁵ 1.0 × 10⁻⁵ 36 *Upper limit reportedbecause off-rate is slower than the limit of detection, which isapproximately 5 × 10⁻⁶ s⁻¹.

TABLE 8 NEG276-LALA dissociation constants (K_(D)) determined byForteBio kinetic binding assays ANGPTL4 Average K_(D) Antibody Proteink_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (pM) (pM) NEG276- Human ANGPTL4(26-406)4.1 × 10⁵ 5.9 × 10⁻⁶ 14 13 LALA 3.6 × 10⁵ 6.0 × 10⁻⁶ 17 4.3 × 10⁵ 4.9 ×10⁻⁷ ≦12*  6.8 × 10⁵ 7.0 × 10⁻⁶ 10 Cyno ANGPTL4(26-406) 3.6 × 10⁵ 5.4 ×10⁻⁶ 15 15 3.7 × 10⁵ 4.1 × 10⁻⁶ ≦14*  Rat ANGPTL4(24-405) 1.6 × 10⁵ 1.2× 10⁻³ 7470  6343  1.7 × 10⁵ 1.1 × 10⁻³ 6030  5.2 × 10⁴ 2.8 × 10⁻⁵ 5530 Mouse ANGPTL4(26-410) 3.6 × 10⁵ 2.9 × 10⁻³ 8250  6200  4.3 × 10⁵ 1.8 ×10⁻³ 4150  Human ANGPTL4(26-161) 2.2 × 10⁵ 3.1 × 10⁻⁶ ≦23*  38 1.8 × 10⁵9.2 × 10⁻⁶ 52 Cyno ANGPTL4(26-161) 2.7 × 10⁵ 6.4 × 10⁻⁶ 24 56 1.9 × 10⁵1.7 × 10⁻⁵ 87 Human ANGPTL3(17-460) —* —* >6000**  >6000**  —*—* >6000**  *Upper limit reported because the dissociation rate isslower that the limit of detection, which is approximately 5 × 10⁻⁶ s⁻¹.**No binding was detected at the highest concentration of antibodytested, 25 nM.

Example 12: Epitope Mapping by Hydrogen-Deuterium Exchange/MassSpectrometry

Hydrogen-deuterium exchange (HDx) in combination with mass spectrometry(MS) (Woods, 2001) was used to map the binding site of antibodies NEG276and NEG318 on the ANGPTL4 N-terminal domain. In HDx, exchangeable amidehydrogens of proteins are replaced by deuterium. This process issensitive to protein structure/dynamics and solvent accessibility and,therefore, able to report on ligand binding. The goal of theseexperiments was the identification of the epitopes of NEG276 and NEG318on ANGPTL4.

Automated HDx/MS experiments were performed using methods similar tothose described in the literature (Chalmers, 2006). The experiments wereperformed on a Waters HDx-MS platform, which includes a LEAPautosampler, nanoACQUITY UPLC System, and Synapt G2 mass spectrometer.The deuterium buffer used to label the protein backbone with deuteriumwas 50 mM D-Tris-HCl (pH 7.4), 500 mM NaCl, 15% glycerol, and 0.1%n-octyl β-D-maltoside; the overall percentage of deuterium in thesolution was 82.5%. For human ANGPTL4(26-161) deuterium labelingexperiments in the absence of ANGPTL4 antibody, 300 pmol of humanANGPLT4(26-161) (1.3 μl) was diluted using 100 μl of the deuteriumbuffer in a chilled tube and incubated for 25 minutes on a rotator at 4°C. The labeling reaction was then quenched with 100 μl of chilled quenchbuffer on ice for three minutes. After three minutes, the quenchedsolution was injected onto the LC-MS system for automated pepsindigestion and peptide analysis. For human ANGPTL4(26-161) deuteriumlabeling experiments in the presence of bound ANGPTL4 antibody, 300 pmolof the ANGPTL4 antibody was first immobilized on Thermo Protein G Plusbeads and cross-linked using disuccinimidyl suberate (DSS). To performthe labeling experiments, the antibody beads (containing 300 pmolantibody) were incubated with 300 pmol human ANGPTL4(26-161) for 30minutes at 4° C. After 30 minutes the beads were washed with 200 μl ofTris buffer. Then 200 μl of chilled deuterium buffer was added and thecomplex was incubated for 25 minutes at 4° C. After 25 minutes, thelabeling reaction was quenched with 125 μl of chilled quench buffer onice for 2.5 minutes. After spinning the sample for 30 seconds in acentrifuge, the quenched solution was injected onto the LC-MS system forautomated pepsin digestion and peptide analysis.

All measurements were carried out using a minimum of three analyticaltriplicates. All deuterium exchange experiments were quenched using 0.5M TCEP and 3 M urea (pH 2.5). After quenching, the exchanged antigen wassubjected to on-line pepsin digestion using a Poroszyme ImmobilizedPepsin column (2.1×30 mm) at 12° C. followed by trapping on a WatersVanguard HSS T3 trapping column. Peptides were eluted from the trappingcolumn and separated on a Waters CSH C18 1×100 mm column (maintained at1° C.) at a flow rate of 40 μl/min using a binary eight minute gradientof 2 to 35% B (mobile phase A was 99.9% water and 0.1% formic acid;mobile phase B was 99.9% acetonitrile and 0.1% formic acid).

In these deuterium exchange experiments, peptides covering 87% of theANGPTL4 N-terminal domain sequence were detected. The detected peptides,and the reduction in deuterium incorporation for each peptide, areindicated in Table 9. The HDxMS mapping experiment identified threeregions of the ANGPTL4 N-terminal domain that were significantlyprotected by both NEG276 and NEG318: amino acids 26-35 (G₂₆PVQSKSPRF₃₅)(SEQ ID NO: 165), amino acids 42-68 (N₄₂VLAHGLLQLGQGLREHAERTRSQLSA₆₈)(SEQ ID NO: 174) and amino acids 69-95 (L₆₉ERRLSACGSACQTEGSTDLPLAPES₉₅)(SEQ ID NO: 190). The observation that NEG276 and NEG318 protectmultiple regions of the ANGPTL4 N-terminal domain from deuteriumincorporation is consistent with results from linear peptide epitopemapping which suggest that NEG276 and NEG318 have conformational ratherthan linear epitopes (see Example 13).

TABLE 9 Effect of NEG276 and NEG318 binding on deuteriumincorporation into human ANGPLT4(26-161). Foreach peptide detected by mass spectrometry, thereduction in deuterium incorporation (in Daltons)for the antibody/ANGPTL4 complex relative to ANGPTL4 alone is shown.Reduction  in deuterium incorpor- SEQ ation Peptide ID (Daltons) NameSequence NO NEG276 NEG318 26-35 GPVQSKSPRF 165 1.1 1.2 28-38 VQSKSPRFASW166 1.0 0.9 42-49 NVLAHGLL 167 <0.5 <0.5 42-51 NVLAHGLLQL 168 <0.5 <0.544-51 LAHGLLQL 169 <0.5 <0.5 42-54 NVLAHGLLQLGQG 170 0.8 0.8 42-55NVLAHGLLQLGQGL 171 0.9 0.8 42-57 NVLAHGLLQLGQGLRE 172 0.9 0.9 42-66NVLAHGLLQLGQGLREHAERTRSQL 173 1.2 1.6 42-68 NVLAHGLLQLGQGLREHAERTRSQL174 1.3 1.8 SA 44-66 LAHGLLQLGQGLREHAERTRSQL 175 1.1 1.6 45-57AHGLLQLGQGLRE 176 0.7 0.9 45-66 AHGLLQLGQGLREHAERTRSQL 177 1.0 1.5 49-66LQLGQGLREHAERTRSQL 178 0.5 1.0 52-66 GQGLREHAERTRSQL 179 <0.5 0.9 69-102 LERRLSACGSACQTEGSTDLPAPES 180 1.4 1.9 RVDPEVL  76-102CGSACQTEGSTDLPAPESRVDPEVL 181 1.0 1.2  86-102 STDLPAPESRVDPEVL 182 1.11.1  96-102 RVDPEVL 183 <0.5 <0.5 103-109 HSLQTQL 184 0.5 0.5 110-119KAQNSRIQQL 185 <0.5 <0.5 110-135 KAQNSRIQQLFHKVAQQQRHLEKQH 186 <0.5 <0.5L 120-135 FHKVAQQQRHLEKQHL 187 <0.5 <0.5 141-147 QSQFGLL 188 0.6 0.6144-155 FGLLDHKHLDHE 189 <0.5 <0.5

Example 13: Epitope Mapping by Linear Peptide Binding

The ability of selected antibodies of invention, namely NEG276 andNEG318, to bind to linear 15-amino-acid peptides derived from theN-terminal coiled coil domain of human ANGPTL4 was tested. A total of 43peptides were synthesized and purified using standard methods; thesequence of the peptides are shown in Table 10. The peptides wereimmobilized on a glass surface, and the ability of NEG276 and NEG318 tobind to the immobilized peptides was evaluated using experimentalmethods optimized for linear peptide epitope mapping at JPT PeptideTechnologies (Berlin, Germany). An antibody that does not bind toANGPTL4 was used as a control for non-specific binding. No specificbinding of NEG276 or NEG318 to any of the 43 15-mer peptides wasobserved in these experiments. These results strongly suggest that theepitopes of NEG276 and NEG318 are not linear ANGPTL4 peptides, butinstead are conformational epitopes.

TABLE 10 Sequences of linear ANGPTL4-derived peptides used for peptide binding  experiments. Name (SEQ PeptideID NO.) GPVQSKSPRFASWDE P1 (191) QSKSPRFASWDEMNV P2 (192)SPRFASWDEMNVLAH P3 (193) FASWDEMNVLAHGLL P4 (194) WDEMNVLAHGLLQLGP5 (195) MNVLAHGLLQLGQGL P6 (196) LAHGLLQLGQGLREH P7 (197)GLLQLGQGLREHAER P8 (198) QLGQGLREHAERTRS P9 (199) QGLREHAERTRSQLSP10 (200) REHAERTRSQLSALE P11 (201) AERTRSQLSALERRL P12 (202)TRSQLSALERRLSAS P13 (203) QLSALERRLSASGSA P14 (204) ALERRLSASGSASQGP15 (205) RRLSASGSASQGTEG P16 (206) SASGSASQGTEGSTD P17 (207)GSASQGTEGSTDLPL P18 (208) SQGTEGSTDLPLAPE P19 (209) TEGSTDLPLAPESRVP20 (210) STDLPLAPESRVDPE P21 (211) LPLAPESRVDPEVLH P22 (212)APESRVDPEVLHSLQ P23 (213) SRVDPEVLHSLQTQL P24 (214) DPEVLHSLQTQLKAQP25 (215) VLHSLQTQLKAQNSR P26 (216) SLQTQLKAQNSRIQQ P27 (217)TQLKAQNSRIQQLFH P28 (218) KAQNSRIQQLFHKVA P29 (219) NSRIQQLFHKVAQQQP30 (220) IQQLFHKVAQQQRHL P31 (221) LFHKVAQQQRHLEKQ P32 (222)KVAQQQRHLEKQHLR P33 (223) QQQRHLEKQHLRIQH P34 (224) RHLEKQHLRIQHLQSP35 (225) EKQHLRIQHLQSQFG P36 (226) HLRIQHLQSQFGLLD P37 (227)IQHLQSQFGLLDHKH P38 (228) LQSQFGLLDHKHLDH P39 (229) QFGLLDHKHLDHEVAP40 (230) LLDHKHLDHEVAKPA P41 (231) HKHLDHEVAKPARRK P42 (232)KHLDHEVAKPARRKR P43 (233)

Example 14: Effect of ANGPTL4 Antibodies of the Invention on PlasmaTriglyceride Concentrations in Human ANGPTL4 Transgenic Mice

A construct to express transgenic human ANGPTL4 in mice was made byinserting the full-length human ANGPTL4 cDNA sequence into thepolylinker region of the pLIVLE6 vector, which contains the humanapolipoprotein E gene promoter and its hepatic control region. ANGPTL4transgenic mice were generated on a C57BL/6J background and bred atNovartis (East Hanover, N.J.). Transgenic mice were tail-clipped at 7days of age and DNA was extracted from the tails using aREDExtract-N-Amp Tissue PCR Kit (Sigma-Aldrich; St. Louis, Mo.; cat#XNATR). The human ANGPTL4 transgene was detected by using primer pairstargeting the pLIVLE6 vector and targeting ANGPTL4 cDNA. Mice werehoused in solid-bottom cages on a rack equipped to automatically providewater ad libitum, maintained on a 12 hr light/dark cycle (6 am to 6 pm),and given standard rodent chow (Harlan-Teklad; Frederick, Md.;cat#8604). The vivarium was maintained between 68-76° F. with 30-70%humidity. Mice were housed with littermates and received food and waterad libitum during the study, except for 4 hr fasts prior to samplecollection.

Animals were fasted for 4 hr and briefly anesthetized for submandibularblood collection to measure baseline plasma triglyceride concentrations.Mice were then injected intraperitoneally (i.p.) with 30 mg/kg antibodydiluted in PBS (10 mL/kg injection volume). Blood was collected after 4hr fasts on days 1, 2, and 5 post-dose to measure plasma triglycerideand total human IgG concentrations. Blood was collected into BDMicrotainer collection/separator tubes with EDTA (Becton, Dickinson, andCompany; Franklin Lakes, N.J., catalog number 365973). Samples werecentrifuged for 10 min at 20,817×g, and plasma was transferred to a 0.2mL Thermo-strip tube (Thermo-Scientific; Pittsburgh, Pa.; cat# AB 0451)and frozen and stored at −80° C.

Plasma triglyceride concentrations were measured using the Triglyceride(GPO) Liquid Reagent set (Pointe Scientific, Canton, Mich., catalognumber T7532-500). Briefly, 300 μL of assay reagent, pre-warmed to 37°C., was added to 5 μL of plasma in a clear, flat-bottom 96-well plate(Thermo Scientific, catalog number 269620). The plate was mixed on aplate shaker for 30 sec and then placed in a 37° C. incubator for 5 min.Following a 20 sec mix, absorbance at 500 nm was measured using aMolecular Devices SPECTRAmax PLUS plate reader. Triglycerideconcentrations were calculated by using a calibration curve generatedusing known quantities of a triglyceride standard (Pointe Scientific,catalog number T7531-STD).

The antibodies NEG276 and NEG318 both reduced plasma triglyceride levelswhen administered to the human ANGPTL4 transgenic mice (FIG. 3).

Example 15: Effects of Administering One of the ANGPTL4 Antibodies ofthe Invention to Obese, Diabetic, Hypertriglyceridemic CynomolgusMonkeys

To evaluate the pharmacokinetic profile and pharmacological effects ofNEG276-LALA, we administered a single, subcutaneous, 3 mg/kg dose tofour hypertriglyceridemic cynomolgus monkeys. The monkeys used in thisstudy had baseline plasma triglyceride levels ranging from 207 mg/dL to2438 mg/dL. At various timepoints over 5 weeks after NEG276-LALA dosing,plasma samples were collected (blood samples were drawn from animalsprior to morning feeding, but the animals were not fasted overnight).

Total NEG276-LALA plasma concentrations were determined by standardmethods. NEG276-LALA reached an average maximum plasma concentration(C_(max)) of 15,536±2281 ng/mL at 3 days post-dose. At day 21 post-dose,the average NEG276-LALA plasma concentration was 2663 ng/mL (FIG. 4).

Plasma TG, total cholesterol, high-density lipoprotein (HDL)cholesterol, total apolipoprotein B (apoB), and apolipoprotein CIII(apoCIII) concentrations were determined using commercially availableassay kits (TG: Triglyceride (GPO) Liquid Reagent set, PointeScientific, catalog number T7532-500; total cholesterol: CholesterolReagent Set, Pointe Scientific, catalog number C7510-500; HDL:Cholesterol Precipitating Reagent from manual HDL reagent kit, Wako,catalog number 431-52501; total ApoB: K-Assay Apo B, Kamiya BiomedicalCompany, catalog number KAI-004; ApoC-III: ApoC-III Assay Reagent,Randox, catalog number LP-3865).

NEG276-LALA administration resulted in a marked decrease in plasmatriglyceride (TG) levels. Peak plasma TG lowering was observed on day 7post-dosing; at this time point plasma TG concentrations were 58% lowerthan baseline plasma TG levels. After peak TG lowering occurred on day 7post-dose, plasma TG concentrations remained suppressed by greater than40% relative to baseline concentrations through day 21 post-dose, thenreturned to baseline (FIG. 5). In addition to its effect on plasma TG,NEG276-LALA administration reduced plasma total cholesterolconcentrations by approximately 30% relative to baseline (FIG. 6) andincreased HDL cholesterol concentrations by more than 20% from baselineon days 7 through 21 post-dosing (FIG. 7). In addition, an approximately30% decrease in plasma total apoB concentrations was observed on days 7through 21 post-dose (FIG. 8), and an approximately 25% decrease inplasma apoC-III concentrations relative to baseline was observed on days7 through 21 (FIG. 9). We also evaluated the effect of NEG276-LALAadministration on lipoprotein-associated triglyceride and cholesterollevels by separating lipoprotein components using standardsize-exclusion chromatography methods. Comparison of lipoproteinprofiling data for pre-dose (day 0) and day 7 post-dose samples showedthat NEG276-LALA administration resulted in marked decreases intriglyceride-rich lipoprotein (TRL) associated cholesterol andtriglyceride concentrations (results from one monkey are shown in FIG.10 and FIG. 11).

INCORPORATION BY REFERENCE

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated herein byreference in their entirety.

EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The foregoingdescription and examples detail certain preferred embodiments of theinvention and describe the best mode contemplated by the inventors. Itwill be appreciated, however, that no matter how detailed the foregoingmay appear in text, the invention may be practiced in many ways and theinvention should be construed in accordance with the appended claims andany equivalents thereof.

1-28. (canceled)
 29. A method of treating an ANGPTL4-disorder, themethod comprising administering to a subject afflicted with anANGPTL4-disorder a therapeutically-effective amount of a pharmaceuticalcomposition comprising an antibody or fragment thereof that bindsspecifically to human ANGPTL4, wherein the antibody or fragment thereofcomprises: a heavy chain variable region comprising a HCDR1 comprisingthe amino acid sequence of SEQ ID NO:7, a HCDR2 comprising the aminoacid sequence of SEQ ID NO:8, and a HCDR3 comprising the amino acidsequence of SEQ ID NO:9, and a light chain variable region comprising aLCDR1 comprising the amino acid sequence of SEQ ID NO:17, a LCDR2comprising the amino acid sequence of SEQ ID NO:18, and a LCDR3comprising the amino acid sequence of SEQ ID NO:19.
 30. The method ofclaim 29, wherein the subject is afflicted with one or more of severehypertriglyceridemia, hypertriglyceridemia associated with obesity, typeV hypertriglyceridemia, and chylomicronemia.
 31. The method of claim 29,wherein the subject is afflicted with one or more of primarydyslipidemia, metabolic syndrome, and type 2 diabetes. 32-33. (canceled)34. The method of claim 29, wherein the antibody or fragment thereof isa monoclonal antibody, humanized antibody, single chain antibody, Fabfragment, Fv fragment, F(ab′)2 fragment, or scFv fragment.
 35. Themethod of claim 29, wherein the antibody or fragment thereof is an IgG1or IgG4 isotype antibody or fragment thereof.
 36. The method of claim29, wherein the antibody or fragment thereof comprises a heavy chainvariable region comprising an amino acid sequence that is at least 90%identical to SEQ ID NO:13, and a light chain variable region comprisingan amino acid sequence that is at least 90% identical to SEQ ID NO:23.37. The method of claim 29, wherein the antibody or fragment thereofcomprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO:13, and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO:23.
 38. The method of claim 29,wherein the antibody or fragment thereof comprises a heavy chaincomprising an amino acid sequence that is at least 90% identical to SEQID NO:15, and a light chain comprising an amino acid sequence that is atleast 90% identical to SEQ ID NO:25.
 39. The method of claim 29, whereinthe antibody or fragment thereof comprises a heavy chain comprising theamino acid sequence of SEQ ID NO:15, and a light chain comprising theamino acid sequence of SEQ ID NO:25.
 40. The method of claim 29, whereinthe antibody or fragment thereof comprises a heavy chain comprising anamino acid sequence that is at least 90% identical to SEQ ID NO:28, anda light chain comprising an amino acid sequence that is at least 90%identical to SEQ ID NO:25.
 41. The method of claim 29, wherein theantibody or fragment thereof comprises a heavy chain comprising theamino acid sequence of SEQ ID NO:28, and a light chain comprising theamino acid sequence of SEQ ID NO:25.
 42. The method of claim 29, whereinthe antibody or fragment thereof is a bispecific antibody of fragmentthereof.
 43. A method of treating an ANGPTL4-disorder, the methodcomprising administering to a subject afflicted with an ANGPTL4-disordera therapeutically-effective amount of a pharmaceutical compositioncomprising an antibody or fragment thereof that binds specifically tohuman ANGPTL4, wherein the antibody or fragment thereof comprises: aheavy chain variable region comprising a HCDR1 comprising the amino acidsequence of SEQ ID NO:10, a HCDR2 comprising the amino acid sequence ofSEQ ID NO:11, and a HCDR3 comprising the amino acid sequence of SEQ IDNO:12; and a light chain variable region comprising a LCDR1 comprisingthe amino acid sequence of SEQ ID NO:20, a LCDR2 comprising the aminoacid sequence of SEQ ID NO:21, and a LCDR3 comprising the amino acidsequence of SEQ ID NO:22.
 44. The method of claim 43, wherein thesubject is afflicted with one or more of severe hypertriglyceridemia,hypertriglyceridemia associated with obesity, type Vhypertriglyceridemia, and chylomicronemia.
 45. The method of claim 43,wherein the subject is afflicted with one or more of primarydyslipidemia, metabolic syndrome, and type 2 diabetes.
 46. The method ofclaim 43, wherein the antibody or fragment thereof is a monoclonalantibody, humanized antibody, single chain antibody, Fab fragment, Fvfragment, F(ab′)2 fragment, or scFv fragment.
 47. The method of claim43, wherein the antibody or fragment thereof is an IgG1 or IgG4 isotypeantibody or fragment thereof.
 48. The method of claim 43, wherein theantibody or fragment thereof comprises a heavy chain variable regioncomprising an amino acid sequence that is at least 90% identical to SEQID NO:13, and a light chain variable region comprising an amino acidsequence that is at least 90% identical to SEQ ID NO:23.
 49. The methodof claim 43, wherein the antibody or fragment thereof comprises a heavychain variable region comprising the amino acid sequence of SEQ IDNO:13, and a light chain variable region comprising the amino acidsequence of SEQ ID NO:23.
 50. The method of claim 43, wherein theantibody or fragment thereof comprises a heavy chain comprising an aminoacid sequence that is at least 90% identical to SEQ ID NO:15, and alight chain comprising an amino acid sequence that is at least 90%identical to SEQ ID NO:25.
 51. The method of claim 43, wherein theantibody or fragment thereof comprises a heavy chain comprising theamino acid sequence of SEQ ID NO:15, and a light chain comprising theamino acid sequence of SEQ ID NO:25.
 52. The method of claim 43, whereinthe antibody or fragment thereof comprises a heavy chain comprising anamino acid sequence that is at least 90% identical to SEQ ID NO:28, anda light chain comprising an amino acid sequence that is at least 90%identical to SEQ ID NO:25.
 53. The method of claim 43, wherein theantibody or fragment thereof comprises a heavy chain comprising theamino acid sequence of SEQ ID NO:28, and a light chain comprising theamino acid sequence of SEQ ID NO:25.
 54. The method of claim 43, whereinthe antibody or fragment thereof is a bispecific antibody of fragmentthereof.